Hydrophilic polymeric particles and methods for making and using same

ABSTRACT

A method of forming a particle includes, in a disperse phase within an aqueous suspension, polymerizing a plurality of mer units of a hydrophilic monomer having a hydrophobic protection group, thereby forming a polymeric particle including a plurality of the hydrophobic protection groups. The method further includes converting the polymeric particle to a hydrophilic particle.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a continuation of U.S. patent application Ser. No.13/762,941, entitled “Hydrophilic polymeric particles and methods formaking and using same,” and filed Feb. 8, 2013, which claims benefit ofU.S. Provisional Application No. 61/597,053, filed Feb. 9, 2012, claimsbenefit of U.S. Provisional Application No. 61/719,045, filed Oct. 26,2012, and claims benefit of U.S. Provisional Application No. 61/731,873,filed Nov. 30, 2012, each of which is incorporated herein by referencein its entirety.

FIELD OF THE DISCLOSURE

This disclosure, in general, relates to hydrophilic polymeric particlesand relates to methods for making and using such hydrophilic polymericparticles.

BACKGROUND

Polymeric particles are increasingly being used as components inseparation techniques and to assist with detecting analytes in bothchemical and biological systems. For example, polymeric particles havebeen used in chromatographic techniques to separate target moleculesfrom a solution. In another example, polymeric particles having amagnetic coating are utilized in magnetic separation techniques. Morerecently, polymeric particles have been used to enhance ELISA-typetechniques and can be used to capture polynucleotides.

Nevertheless, such separation and analytical techniques have suffered asa result of variance in particle size. Large variance in particle sizeleads to variance in particle weight, as well as variance in the numberof reaction sites available for interacting with target analytes. Formagnetic separations techniques, variance in size can lead to lowefficiency separations. For chromatographic techniques and variouspolynucleotide capture techniques, variance in size can lead to variancein the number of sites available for interacting with polynucleotides,leading to variance in capture or separation efficiency.

As such, an improved polymeric particle and method for manufacturingsuch a polymeric particle would be desirable.

SUMMARY

In a first aspect, a method of forming a particle includes, in adisperse phase within an aqueous suspension, polymerizing a plurality ofmer units of a hydrophilic monomer having a hydrophobic protectiongroup, thereby forming a polymeric particle including a plurality of thehydrophobic protection groups, and converting the polymeric particle toa hydrogel particle.

In a second aspect, a method of forming a particle includes, in adisperse phase within an aqueous suspension, polymerizing a plurality ofmer units of an acrylamide monomer having a hydrophobic protectiongroup, thereby forming a polymeric particle including a plurality of thehydrophobic protection groups, and converting the polymeric particle toa hydrophilic particle.

In a third aspect, a method of forming a particle includes, in adisperse phase within an aqueous suspension, polymerizing a plurality ofmer units of an radically polymerizable monomer with a diacrylamidecrosslinker having a hydrophobic protection group, thereby forming apolymeric particle including a plurality of the hydrophobic protectiongroups. The method further includes removing at least a portion ofplurality of the hydrophobic protection groups.

In a fourth aspect, a method of forming a particle includes polymerizinga plurality of mer units of a hydrophilic monomer having a hydrophobicprotection group, thereby forming a polymeric particle including aplurality of the hydrophobic protection groups; removing at least aportion of plurality of the hydrophobic protection groups from thepolymeric particle to form a hydrophilic particle; and binding anoligonucleotide to the hydrophilic particle

In a fifth aspect, a plurality of particles includes at least 100,000particles. At least one particle of the plurality of particles includesa hydrogel. The plurality of particles has an average particle size ofnot greater than 100 micrometers and a coefficient of variance of notgreater than 5%.

In a sixth aspect, a system includes an array of wells. At least onewell of the array of wells is operatively connected with an ISFETsensor. The system further includes a plurality of hydrogel particleshaving a coefficient of variance of not greater than 5%. At least one ofthe hydrogel particles of the plurality of hydrogel particles isdisposed in a well of the array of wells.

In a seventh aspect, a plurality of particles is formed by the methodincluding, in a disperse phase within an aqueous suspension,polymerizing a plurality of mer units of a hydrophilic monomer having ahydrophobic protection group, thereby forming a polymeric particleincluding a plurality of the hydrophobic protection groups, andincluding converting the polymeric particle to a hydrogel particle.

In an eighth aspect, a composition includes an aqueous mixture of anacrylamide monomer and a crosslinker, the acrylamide monomer including ahydrophobic protection group, the monomer and crosslinker included in amass ratio of monomer:crosslinker in a range of 15:1 to 1:2.

In a ninth aspect, a method of sequencing a polynucleotide includesproviding a device including an array of wells. At least one well isoperatively connected to an ISFET and includes a particle formed by themethod of the above aspects. The particle is attached to apolynucleotide. The method further includes applying a solutionincluding nucleotides of a predetermined type to the device andobserving an ionic response to the applying the solution.

In a tenth aspect, a method for nucleotide incorporation includesproviding a particle formed by the method of the above aspects. Theparticle is attached to a nucleic acid duplex including a templatenucleic acid hybridized to a primer. The duplex is bound to apolymerase. The method further includes contacting the particle with oneor more nucleotides and incorporating at least one nucleotide onto theend of the primer using the polymerase.

In an eleventh aspect, a method of forming a particle includes promotinga seed particle to form a disperse phase in an aqueous suspension, inthe disperse phase, polymerizing a plurality of mer units of ahydrophilic monomer having a hydrophobic protection group, therebyforming a polymeric particle including a plurality of hydrophobicprotection groups, and converting the polymeric particle to a hydrogelparticle.

In a twelfth aspect, a method of forming a particle includes providing aseed particle in an aqueous suspension, the seed particle comprising ahydrophobic polymer, and includes promoting the seed particle to form adisperse phase in the aqueous suspension. The method further includes,in the disperse phase, polymerizing a plurality of mer units of ahydrophilic monomer having a hydrophobic protection group, therebyforming a polymeric particle including a hydrophilic polymer having aplurality of the hydrophobic protection groups. The polymeric particleincludes the hydrophobic polymer. The method also includes cleaving theplurality of hydrophobic protection groups from the hydrophilic polymerand extracting the hydrophobic polymer from the polymeric particle toform a hydrogel particle.

In a thirteenth aspect, a particle includes a polymer formed frompolymerization of hydroxyalkyl acrylamide and a diacrylamide. Thediacrylamide includes a hydroxyl group. The particle absorbs at least300 wt % water based on the weight of the polymer when exposed to water.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure may be better understood, and its numerousfeatures and advantages made apparent to those skilled in the art byreferencing the accompanying drawings.

FIG. 1 includes an illustration of an exemplary process flow formanufacturing an exemplary polymeric particle.

FIG. 2 includes an illustration of an exemplary sequencing methodutilizing polymeric particles.

The use of the same reference symbols in different drawings indicatessimilar or identical items.

DESCRIPTION OF THE PREFERRED EMBODIMENT(S)

In an exemplary embodiment, a method of forming polymeric particlesincludes polymerizing, in a dispersed phase within an aqueoussuspension, a plurality of mer units of a monomer having hydrophilicfunctional groups protected with a hydrophobic protection group.Polymerizing forms a polymeric particle including a plurality of thehydrophobic protection groups. The method further includes convertingthe polymeric particle to a hydrophilic particle, such as a hydrogelparticle. In an example, the monomer includes a hydrophilic radicallypolymerizable monomer, such as a hydrophilic vinyl-based monomer, inparticular an acrylamide. The monomer is a hydrophilic monomer includinghydrophilic functionality protected with a hydrophobic protection group.For example, the hydrophobic protection group can include silylfunctionality or derivatives thereof. Polymerizing can also includepolymerizing in the presence of a crosslinker, such as a vinylcrosslinker, including exemplary diacrylamide crosslinkers. Thecrosslinkers can be protected crosslinkers having hydrophobic protectiongroups. In an example, converting the polymeric particle to ahydrophilic particle can include removing at least a portion of thehydrophobic protection groups from the polymeric particle. Inparticular, the hydrophobic protection group can be an acid-cleavableprotection group, and removing the hydrophobic protection group caninclude acid cleaving the hydrophobic protection group from thepolymeric particle.

Exemplary polymeric particles made by such methods can have a desirablesize or coefficient of variance. In particular, the polymeric particlescan be hydrophilic. For example, the polymeric particles can includehydrogel particles. Further, the polymeric particles can have an averageparticle size of not greater than 100 μm, such as not greater than 30μm, not greater than 3 μm, or not greater than 2 μm. The polymericparticles can have a coefficient of variance not greater than 15%, suchas not greater than 5%.

In particular, such particles can be useful in capturing targetanalytes, such as polynucleotides. In an example, the polymericparticles can be useful in sequencing polynucleotides using sequencingmethods that involve light detection or sequencing methods that involveion detection.

In a particular embodiment, a dispersed phase is formed within anaqueous suspension. The dispersed phase is preferably hydrophobic. In anexample, the dispersed phase is formed as a result of promoting seedparticles, such as hydrophobic seed particles, to yield the dispersedphase. Promoting facilitates absorption of hydrophobic components in theseed particle.

Monomers having removable hydrophobic protection groups prefer thedispersed phase. The monomers polymerize within the dispersed phase.Optionally, a crosslinker is polymerized with the monomers within thedispersed phase. In an example in which the dispersed phase is formedfrom a seed particle, such as a hydrophobic seed particle, the polymerassociated with the seed particle can be removed. For example, thepolymer of the seed particle can be dissolved using solvents and can beextracted from the polymeric particle.

The hydrophobic protection groups can be removed, such as throughcleaving at least a portion of the hydrophobic protection groups fromthe polymeric particle. As a result, a hydrophilic particle is formed,such as a hydrogel particle.

In an example, the resulting in hydrophilic particle can be activated tofacilitate conjugation with a target analyte, such as a polynucleotide.For example, cleaving the hydrophobic protection groups can leave ahydrophilic functional group, such as hydroxyl groups, amino groups,thiol groups, or a combination thereof, on the hydrophilic particle. Ina particular example, hydroxyl groups can be activated by converting thehydroxyl groups to sulfonate ester groups or chlorine. Sulfonate esterfunctional groups or chlorine can be substituted or replaced usingnucleophilic substitution. In particular, oligonucleotides having anucleophile terminal group, such as an amine or a thiol group, can beattached to the hydrophilic particle by nucleophilic substitution forthe sulfonate groups or chlorine. Such particles can be particularlyuseful in capturing polynucleotides for use in sequencing techniques.

In another example, the sulfonated particles can be further reacted withmono- or multi-functional mono- or multi-nucleophilic reagents that canform an attachment to the particle while maintaining nucleophilicactivity for oligonucleotides comprising electrophilic groups such asmaleimide. In addition, the residual nucleophilic activity can beconverted to electrophilic activity by attachment to reagents comprisingmulti-electrophilic groups, which are subsequently to attach tooligonucleotides comprising nucleophilic groups.

Other conjugation techniques include the use of monomers that comprisehydrophobic protecting groups on carboxylic acids during particlesynthesis. De-protection of the carboxylic acid group makes available acarboxylic acid group that can be further reacted with oligonucleotideshaving a nucleophilic group, such as an amine or causing attachment ofthe oligonucleotide

Other conjugation techniques include the use of monomers that comprisehydrophobic protecting groups on amines during particle synthesis.De-protection of the amine group makes available a nucleophilic groupthat can be further modified with amine reactive bi-functionalbis-electrophilic reagents that yield a mono-functional electrophilicgroup subsequent to attachment to the polymer particle. Such anelectrophilic group can be reacted with oligonucleotides having anucleophilic group, such as an amine or thiol, causing attachment of theoligonucleotide by reaction with the vacant electrophile.

As illustrated in FIG. 1, a method 100 includes providing a seedparticle 102. Monomers are added to the suspension and preferably residein the dispersed phase 104 formed from a promoted seed particle. Themonomer and optionally, a crosslinker are polymerized to form apolymeric particle 108. The polymeric particle 108 can be stripped ofthe seed polymer to form the polymeric particle 110. The hydrophobicprotection groups on the polymeric particle 110 are removed to form ahydrophilic particle 112. The hydrophilic particle 112 can be activatedto form a conjugated particle 114.

The seed particle 102 can include a seed polymer. In an example, theseed polymer is hydrophobic. In particular, the seed polymer can includea styrenic polymer, an acrylic polymer, an acrylamide, anotherhydrophobic vinyl polymer, or any combination thereof. In an example,the seed particle 102 is monodisperse, for example, having a coefficientof variance of not greater than 20%. Coefficient of variance (CV) isdefined as 100 times the standard deviation divided by the average,where “average” is mean particle diameter and standard deviation isstandard deviation in particle size. Alternatively, the “average” can beeither the z-average or mode particle diameter. In accordance with usualpractice, CV is calculated on the main mode, i.e. the main peak, therebyexcluding minor peaks relating to aggregates. Thus some particles belowor above mode size may be discounted in the calculation which may, forexample, be based on about 90% of total particle number of detectableparticles. Such a determination of CV is performable on a CPS disccentrifuge. In particular, a population of seed particles 102 can have acoefficient of variance of not greater than 10%, such as not greaterthan 5.0%, not greater than 3.5%, not greater than 3%, not greater than2.5%, not greater than 2%, or even not greater than 1.0%. Further, theseed particle 102 can have an initial particle size of not greater than0.6 μm. For example, the initial particle size can be not greater than0.45 μm, such as not greater than 0.35 μm, or even not greater than 0.15μm. Alternatively, larger seed particles having an initial particle sizeof at least 3 μm, such as at least 5 μm, at least 10 μm, at least 20 μm,or at least 50 μm, can be used to form larger polymeric particles. In anexample, the initial particle size can be not greater than 100 μm.

The seed particle 102 can be promoted within an aqueous suspension toform a promoted dispersed phase 104. In particular, promoting the seedparticles includes mixing a solvent and a promoter with the seedparticle within the aqueous suspension to form the dispersed phase.Promoted seed particles more readily absorb hydrophobic components. Thesolvent can be water-miscible. For example, the solvent can include analdehyde or ketone, such as formaldehyde, acetone, methyl ethyl ketone,diisopropyl ketone, dimethyl formamide, or combinations thereof; anether solvent, such as tetrahydrofuran, dimethyl ether, or combinationsthereof; an ester solvent; a heterocyclic solvent, such as pyridine,dioxane, tetrahydrofurfuryl alcohol, N-methyl-2-pyrrolidone, orcombinations thereof; or combinations thereof. In an example, thesolvent can include a ketone, such as acetone. In another example, thesolvent can include an ether solvent, such as tetrahydrofuran. In anadditional example, the solvent can include a heterocyclic solvent, suchas pyridine.

The promoter or promoting agent can be hydrophobic and have a low watersolubility, such as a water solubility of not greater than 0.01 g/l at25° C. For example, the promoter can include dioctanoyl peroxide,dioctyladipate, n-butyl phthalate, dodecanol, polystyrene with molecularweight below 20 kD, or a combination thereof. In an example, thedioctanoyl peroxide can also perform as an initiator for apolymerization reaction. The promoter can also be a low molecular weightpolystyrene, for example, made in a separate polymerization step using alow monomer/initiator ratio or the addition of chain transfer reagentsduring the seed polymerization. The promoter is typically emulsified ina high pressure homogenizer.

The aqueous suspension can also include a surfactant. The surfactant canbe an ionic surfactant, an amphoteric surfactant, or a non-ionicsurfactant. The ionic surfactant can be an anionic surfactant. Inanother example, the ionic surfactant can be a cationic surfactant. Anexemplary anionic surfactant includes a sulfate surfactant, a sulfonatesurfactant, a phosphate surfactant, a carboxylate surfactant, or anycombination thereof. An exemplary sulfate surfactant includes alkylsulfates, such as ammonium lauryl sulfate, sodium lauryl sulfate (sodiumdodecyl sulfate, (SDS)), or a combination thereof; an alkyl ethersulfate, such as sodium laureth sulfate, sodium myreth sulfate, or anycombination thereof; or any combination thereof. An exemplary sulfonatesurfactant includes an alkyl sulfonate, such as sodium dodecylsulfonate; docusates such as dioctyl sodium sulfosuccinate; alkyl benzylsulfonate; or any combination thereof. An exemplary phosphate surfactantincludes alkyl aryl ether phosphate, alkyl ether phosphate, or anycombination thereof. An exemplary carboxylic acid surfactant includesalkyl carboxylates, such as fatty acid salts or sodium stearate; sodiumlauroyl sarcosinate; a bile acid salt, such as sodium deoxycholate; orany combination thereof.

An exemplary cationic surfactant includes primary, secondary or tertiaryamines, quaternary ammonium surfactants, or any combination thereof. Anexemplary quaternary ammonium surfactant includes alkyltrimethylammoniumsalts such as cetyl trimethylammonium bromide (CTAB) or cetyltrimethylammonium chloride (CTAC); cetylpyridinium chloride (CPC);polyethoxylated tallow amine (POEA); benzalkonium chloride (BAC);benzethonium chloride (BZT); 5-bromo-5-nitro-1,3-dioxane;dimethyldioctadecylammonium chloride; dioctadecyldimethylammoniumbromide (DODAB); or any combination thereof.

An exemplary amphoteric surfactant includes a primary, secondary, ortertiary amine or a quaternary ammonium cation with a sulfonate,carboxylate, or phosphate anion. An exemplary sulfonate amphotericsurfactant includes(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate); a sultainesuch as cocamidopropyl hydroxysultaine; or any combination thereof. Anexemplary carboxylic acid amphoteric surfactant includes amino acids,imino acids, betaines such as cocamidopropyl betaine, or any combinationthereof. An exemplary phosphate amphoteric surfactant includes lecithin.In a further example, the surfactant can be a non-ionic surfactant suchas a polyethylene glycol-based surfactant.

Returning to FIG. 1, monomers added to suspension preferably naturallyreside in the dispersed phase 104 formed from a promoted seed particle.A crosslinker, such as a hydrophobic crosslinker can also be added tothe aqueous suspension and preferentially can reside in the dispersedphase. In an example, the crosslinker has a water solubility of notgreater than 10 g/l. Further, a porogen can be added to the aqueoussuspension and preferentially can reside within the dispersed phase. Ina further example, the dispersed phase can include acryditeoligonucleotides, such as an ion-exchanged acrydite oligonucleotide. Asillustrated in FIG. 1, the monomer and optionally, the crosslinker arepolymerized to form a polymeric particle 108.

The monomer can be a radically polymerizable monomer such as avinyl-based monomer. In particular, the monomer can include ahydrophilic monomer coupled to a hydrophobic protection group. In anexample, the hydrophilic monomer can include acrylamide, vinyl acetate,hydroxyalkylmethacrylate, or any combination thereof. In a particularexample, the hydrophilic monomer is an acrylamide, such as an acrylamideincluding hydroxyl groups, amino groups, carboxyl groups, or acombination thereof. In an example, the hydrophilic monomer is anaminoalkyl acrylamide, an acrylamide functionalized with an amineterminated polypropylene glycol (C, illustrated below), anacrylopiperazine (D, illustrated below), or a combination thereof. Inanother example, the acrylamide can be a hydroxyalkyl acrylamide, suchas hydroxyethyl acrylamide. In particular, the hydroxyalkyl acrylamidecan include N-tris(hydroxymethyl)methyl)acrylamide (A, illustratedbelow), N-(hydroxymethyl)acrylamide (B, illustrated below), or acombination thereof. In a further example, a mixture of monomers, suchas a mixture of hydroxyalky acrylamide and amine functionalizeacrylamide or a mixture of acrylamide and amine functionalizedacrylamide, can be used. In an example, the amine functionalizeacrylamide can be included in a ratio of hydroxyalkyl acrylamide:aminefunctionalized acrylamide or acrylamide:amine functionalized acrylamidein a range of 100:1 to 1:1, such as a range of 100:1 to 2:1, a range of50:1 to 3:1, a range of 50:1 to 5:1 or even a range of 50:1 to 10:1.

In a particular example, the hydrophilic monomer includes hydroxylgroups or includes amines. The hydrophobic protection group shields thehydrophilicity of the monomer, for example, by bonding to a hydroxylgroup or an amine group. Such protection groups are referred to hereinas hydroxyl or hydroxy protection groups when bonding to a hydroxylgroup. In particular, the hydrophobic protection group is removable,such as through cleaving, for example, acid cleaving. The hydrophobicgroup can be selected to cleave under acidic conditions that do notresult in the hydrolysis of the underlying polymer or portions thereof.For example for pH values lower than 6, when an acrylamide polymer ispresent, the hydrophobic protection group cleaves at a pH higher than apH at which the amide portion of the acrylamide hydrolyzes. For pHvalues higher than 9, the hydrophobic protection group cleaves at a pHlower than a pH at which the amide portion of the acrylamide hydrolyzes.

An exemplary hydrophobic protection group includes an organometallicmoiety. For example, the organometallic moiety can form a silyl etherfunctional group. The silyl ether functional group can be derived from ahalogenated silyl compound, such as a compound of the generalformulation R₁Si(R₂)(R₃)(R₄), wherein R₁ is a halogen, such as chlorineand R₂, R₃, and R₄ are independently selected from hydrogen, alkylgroups such as methyl, ethyl, propyl, butyl, aryl group, silyl groups,ether derivatives thereof, or any combination thereof. An exemplarysilyl ether functional group is derived from tert-butyldimethylsilylchloride, trimethylsilyl chloride, triethylsilyl chloride,tripropylsilyl chloride, tributylsilyl chloride, diphenyl methyl silylchloride, chloro(dimethyl)phenyl silane, or a combination thereof. In aparticular example, the protected monomer includesN-(2-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide or tBDMS-HEAM,N-(2-((triethylsilyl)oxy)ethyl)acrylamide or TES-HEAM, or a combinationthereof. In another example, the hydrophobic protection group caninclude an organic moiety. An exemplary organic moiety can include analkyloxycarbonyl group moiety, such as t-butyloxycarbonyl,fluorenylmethyloxycarbonyl, or a combination thereof. In an example,such an organic moiety can be a hydrophobic protection group bound to anamine functional group, such as an amine functional group of an aminefunctionalized acrylamide or copolymer thereof.

The protected monomer can be included in an amount relative to theinitial seed polymer, expressed as a ratio of weights (protectedmonomer:seed polymer), in a range of 100:1 to 1:2, such as a range of50:1 to 1:1, a range of 45:1 to 2:1, a range of 30:1 to 5:1, or even arange of 20:1 to 8:1. Alternatively, the monomer can be included in anamount in a range of 10:1 to 1:2, such as a range of 5:1 to 1:2, or evena range of 2:1 to 1:2.

The dispersed phase can also include a crosslinker. In an example, thecrosslinker is included in a mass ratio of protected monomer tocrosslinker in a range of 15:1 to 1:2, such as a range of 10:1 to 1:1, arange of 6:1 to 1:1, or even a range of 4:1 to 1:1. The crosslinker canhave a low water solubility (e.g., less than 10 g/l), resulting in apreference for the dispersed phase. In particular, the crosslinker canbe a divinyl crosslinker. For example, a divinyl crosslinker can includea diacrylamide, such as N,N′-(ethane-1,2-diyl)bis(2-hydroxylethyl)acrylamide, N,N′-(2-hydroxypropane-1,3-diyl)diacrylamide, or acombination thereof. In another example, a divinyl crosslinker includesethyleneglycol dimethacrylate, divinylbenzene, hexamethylenebisacrylamide, trimethylolpropane trimethacrylate, a protectedderivative thereof, or a combination thereof. In a further example, thecrosslinker can be protected with a hydrophobic protection group, suchas a hydroxyl protection group. In particular, the hydrophobicprotection group can be an organometallic moiety. For example, theorganometallic moiety can form a silyl ether functional group. Anexemplary silyl ether functional group can be derived fromtert-butyldimethylsilyl chloride, trimethylsilyl chloride, triethylsilylchloride, tripropylsilyl chloride, tributylsilyl chloride, diphenylmethyl silyl chloride, chloro(dimethyl)phenylsilane, or a combinationthereof. An exemplary protected diacrylamide crosslinker includesN,N′-(ethane-1,2-diyl)bis(N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide,N,N′-(N-(2-((tert-butyldimethylsilyl)oxy)propane-1,3-diyl)diacrylamide,N,N′-(ethane-1,2-diyl)bis(N-(2-((triethylsilyl)oxy)ethyl)acrylamide,N,N′-(N-(2-((triethylsilyl)oxy)propane-1,3-diyl)diacrylamide,silyl-protected N-[2-(acryloylamino)-1,2-dihydroxyethyl]acrylamide suchas N,N′(2,3-bis((triethylsilyl)oxy)butane-1,4-diyl)diacrylamide, or acombination thereof. In another example, the protection group caninclude an alkyloxycarbonyl group moiety, such as t-butyloxycarbonyl,fluorenylmethyloxycarbonyl, or a combination thereof. In particular, acrosslinker including a hydroxyl group can be protected with aprotection group, such as those described above in relation to theprotected monomer.

In addition, polymerizing the hydrophilic monomer having a hydrophobicprotection can include polymerizing in the presence of a porogen. Anexemplary porogen includes an aromatic porogen. In example, the aromaticporogen includes benzene, toluene, xylene, mesitylene, phenethylacetate,diethyladipate, hexylacetate, ethylbenzoate, phenylacetate,butylacetate, or a combination thereof. The porogen typically has aSolubility parameter of 15-20. In another example, the porogen is analkanol porogen, such as dodecanol. The porogen can be included inamounts relative to the organic phase within the reactive system in arange of 1 wt % to 99 wt %, such as a range of 30 wt % to 90 wt % oreven a range of 50 wt % to 85 wt %.

The monomer is chosen from a group of monomers that produce a hydrogelin its unprotected form such that oligonucleotides and polymerases canreach their targets during use.

Hydrophilic acrylamides and especially diacrylamides are difficult todissolve in a solvent that is not miscible with water and at the sametime dissolves a hydrophobic seed polymer. The protection group for bothmonomer and crosslinker can be chosen such that the solubility of themonomers in a hydrophobic phase is large enough to achieve aconcentration large enough to carry out the polymerization. At the sametime the protection group may not be so large that polymerization cannotbe carried out because of steric hindrance. The deprotection can beperformed at conditions that will not hydrolyze the polymer.

Optionally, a polymerization initiator can be included. An exemplarypolymerization initiator can initiate polymerization through freeradical generation. An exemplary polymerization initiator includes anazo initiator such as oil soluble azo initiators. Another initiator caninclude ammonium persulfate. A further exemplary initiator can includetetramethylethylenediamine. In an example, the polymerization initiatorcan be included in an amount of 0.001 wt % to 3 wt % based on the weightof the dispersed phase.

Following polymerization, the polymeric particle 108 can be stripped ofthe seed polymer to form the polymeric particle 110 still having thehydrophobic protection groups. For example, the seed polymer can beextracted using a solvent, such as an aldehyde or ketone, such asacetone, methyl ethyl ketone, diisopropyl ketone, butylacetate,cyclohexanone, dimethyl formamide, or a combination thereof; a phthalatesolvent, such as, n-butyl phthalate; an ether solvent, such astetrahydrofuran, diisopropyl ether, methyl tertbutyl ether, dimethylether, diethyl ether, or a combination thereof; an ester solvent, suchas ethyl acetate, butyl acetate, or a combination thereof; aheterocyclic solvent, such as pyridine, dioxane, tetrahydrofurfurylalcohol, or a combination thereof; halogenated solvents such as dichloromethane, chloroform or a combination thereof. Alternatively, the seedpolymer can be extracted following the conversion of the polymericparticle to a hydrophilic particle. For example, the seed polymer can beextracted following deprotecting the polymer of particle, such asremoving the silyl groups on the polymer resulting from the protectedmonomer.

As illustrated in FIG. 1, the polymeric particle 110, once the seedpolymer is extracted, can be converted to a hydrophilic polymericparticle by removing at least a portion of the hydrophobic protectiongroups. For example, the hydrophobic protection groups can beacid-cleaved from the polymeric particles. In particular, such removingcan remove substantially all of the hydrophobic protection groups fromthe polymeric particle, such as removing at least 80% of the hydrophobicprotection groups, or even at least 90% of the hydrophobic protectiongroups.

In an example, the hydrophobic protection groups are acid-cleavedthrough the addition of an acid, such as an organic acid. In particular,the organic acid can have a pKa in a range of 3.0 to 5.5. For example,the organic acid can include acetic acid, lactic acid, citric acid, orany combination thereof. Alternatively, inorganic acids can be used.

Once at least a portion of the hydrophobic protection groups is removed,a hydrophilic particle 112 is formed. The hydrophilic particle 112 canbe a hydrogel particle. A hydrogel is a polymer that can absorb at least20% of its weight in water, such as at least 45%, at least 65%, at least85%, at least 100%, at least 300%, at least 1000%, at least 1500% oreven at least 2000% of its weight in water.

The hydrophilic polymer 112 can be activated to facilitate conjugationwith a target analyte, such as a polynucleotide. For example, functionalgroups on the hydrophilic particle 112 can be enhanced to permit bindingwith target analytes or analyte receptors. In a particular example,functional groups of the hydrophilic polymer can be modified withreagents capable of converting the hydrophilic polymer functional groupsto reactive moieties that can undergo nucleophilic or electrophilicsubstitution. For example, hydroxyl groups on the hydrophilic particle112 can be activated by replacing at least a portion of the hydroxylgroups with a sulfonate group or chlorine. Exemplary sulfonate groupscan be derived from tresyl, mesyl, tosyl, or fosyl chloride, or anycombination thereof. Sulfonate can act to permit nucleophiles to replacethe sulfonate. The sulfonate may further react with liberated chlorineto provide a chlorinated groups that can be used in a process toconjugate the particles. In another example, amine groups on thehydrophilic polymer 112 can be activated.

For example, target analyte or analyte receptors can bind to thehydrophilic polymer through nucleophilic substitution with the sulfonategroup. In particular example, target analyte receptors terminated with anucleophile, such as an amine or a thiol, can undergo nucleophilicsubstitution to replace the sulfonate groups on the surface of thehydrophilic polymer 112. As a result of the activation, a conjugatedparticle 114 can be formed.

In another example, the sulfonated particles can be further reacted withmono- or multi-functional mono- or multi-nucleophilic reagents that canform an attachment to the particle while maintaining nucleophilicactivity for oligonucleotides comprising electrophilic groups, such asmaleimide. In addition, the residual nucleophilic activity can beconverted to electrophilic activity by attachment to reagents comprisingmulti-electrophilic groups, which are subsequently to attach tooligonucleotides comprising nucleophilic groups.

In another example, a monomer containing the functional group can beadded during the polymerization. The monomer can include, for example,an acrylamide containing a carboxylic acid, ester, halogen or otheramine reactive group. The ester group may be hydrolyzed before thereaction with an amine oligo.

Other conjugation techniques include the use of monomers that comprisehydrophobic protecting groups on amines during particle synthesis.De-protection of the amine group makes available a nucleophilic groupthat can be further modified with amine reactive bi-functionalbis-electrophilic reagents that yield a mono-functional electrophilicgroup subsequent to attachment to the polymer particle. Such anelectrophilic group can be reacted with oligonucleotides having anucleophilic group, such as an amine or thiol, causing attachment of theoligonucleotide by reaction with the vacant electrophile.

If the particle 112 is prepared from a combination of amino- andhydroxyl-acrylamides, de-protection of the hydrogel particle results ina combination of nucleophilic amino groups and neutral hydroxyl groups.The amino groups can be modified with di-functional bis-electrophilicmoieties, such as a di-isocyanate or bis-NHS ester, resulting in ahydrophilic particle reactive to nucleophiles. An exemplary bis-NHSester includes bis-succinimidyl C2-C12 alkyl esters, such asbis-succinimidyl suberate or bis-succinimidyl glutarate.

Other activation chemistries include incorporating multiple steps toconvert a specified functional group to accommodate specific desiredlinkages. For example, the sulfonate modified hydroxyl group can beconverted into a nucleophilic group through several methods. In anexample, reaction of the sulfonate with azide anion yields an azidesubstituted hydrophilic polymer. The azide can be used directly toconjugate to an acetylene substituted biomolecule via “CLICK” chemistrythat can be performed with or without copper catalysis. Optionally, theazide can be converted to amine by, for example, catalytic reductionwith hydrogen or reduction with an organic phosphine. The resultingamine can then be converted to an electrophilic group with a variety ofreagents, such as di-isocyanates, bis-NHS esters, cyanuric chloride, ora combination thereof. In an example, using di-isocyanates yields a urealinkage between the polymer and a linker that results in a residualisocyanate group that is capable of reacting with an amino substitutedbiomolecule to yield a urea linkage between the linker and thebiomolecule. In another example, using bis-NHS esters yields an amidelinkage between the polymer and the linker and a residual NHS estergroup that is capable of reacting with an amino substituted biomoleculeto yield an amide linkage between the linker and the biomolecule. In afurther example, using cyanuric chloride yields an amino-triazinelinkage between the polymer and the linker and two residualchloro-triazine groups one of which is capable of reacting with an aminosubstituted biomolecule to yield an amino-triazine linkage between thelinker and the biomolecule. Other nucleophilic groups can beincorporated into the particle via sulfonate activation. For example,reaction of sulfonated particles with thiobenzoic acid anion andhydrolysis of the consequent thiobenzoate incorporates a thiol into theparticle which can be subsequently reacted with a maleimide substitutedbiomolecule to yield a thio-succinimide linkage to the biomolecule.Thiol can also be reacted with a bromo-acetyl group.

Alternatively, acrydite oligonucleotides can be used during thepolymerization to incorporate oligonucleotides. An exemplary acryditeoligonucleotide can include an ion-exchanged oligonucleotides.

Covalent linkages of biomolecules onto refractory or polymericsubstrates can be created using electrophilic moieties on the substratecoupled with nucleophilic moieties on the biomolecule or nucleophiliclinkages on the substrate coupled with electrophilic linkages on thebiomolecule. Because of the hydrophilic nature of most commonbiomolecules of interest, the solvent of choice for these couplings iswater or water containing some water soluble organic solvent in order todisperse the biomolecule onto the substrate. In particular,polynucleotides are generally coupled to substrates in water systemsbecause of their poly-anionic nature. Because water competes with thenucleophile for the electrophile by hydrolyzing the electrophile to aninactive moiety for conjugation, aqueous systems generally result in lowyields of coupled product, where the yield is based on the electrophilicportion of the couple. When high yields of electrophilic portion of thereaction couple are desired, high concentrations of the nucleophile arerequired to drive the reaction and mitigate hydrolysis, resulting ininefficient use of the nucleophile. In the case of polynucleic acids,the metal counter ion of the phosphate can be replaced with a lipophiliccounter-ion, in order to help solubilize the biomolecule in polar,non-reactive, non-aqueous solvents. These solvents can include amides orureas such as formamide, N,N-dimethylformamide, acetamide,N,N-dimethylacetamide, hexamethylphosphoramide, pyrrolidone,N-methylpyrrolidone, N,N,N′,N′-tetramethylurea,N,N′-dimethyl-N,N′-trimethyleneurea, or a combination thereof;carbonates such as dimethyl carbonate, propylene carbonate, or acombination thereof; ethers such as tetrahydrofuran; sulfoxides andsulfones such as dimethylsulfoxide, dimethylsulfone, or a combinationthereof; hindered alcohols such as tert-butyl alcohol; or a combinationthereof. Lipophilic cations can include tetraalkylammomiun ortetraarylammonium cations such as tetramethylamonium, tetraethylamonium,tetrapropylamonium, tetrabutylamonium, tetrapentylamonium,tetrahexylamonium, tetraheptylamonium, tetraoctylamonium, and alkyl andaryl mixtures thereof, tetraarylphosphonium cations such astetraphenylphosphonium, tetraalkylarsonium or tetraarylarsonium such astetraphenylarsonium, and trialkylsulfonium cations such astrimethylsulfonium, or a combination thereof. The conversion ofpolynucleic acids into organic solvent soluble materials by exchangingmetal cations with lipophilic cations can be performed by a variety ofstandard cation exchange techniques.

In another example, particles can be formed using an emulsionpolymerization technique in which a hydrophobic phase forms a dispersedphase within a hydrophilic phase. The monomers, crosslinkers, and otheragents and compounds described above that favor hydrophobic phases tendto reside in the hydrophobic phase in which polymerization occurs.

Surfactants, such as those described above can be use in the hydrophilicphase to support emulsion formation. When a seed particle is used, thesurfactant can be used at a concentration below the critical micelleconcentration. Alternatively, the surfactant can be used at aconcentration greater than the critical micelle concentration. Emulsionpolymerization is typically performed with a water soluble initiatorlike potassium or ammonium persulphate.

By adding the intitiator to a heated emulsion of monomers particlenucleation starts in the water phase and the formed particles arestabilized by the surfactants. If most of the particles are createdwithin a short time period, monosized seed particles may be produced.The later increase of particle size happens because the monomer diffusesthrough the water phase from the large monomer droplets to the muchsmaller seed particles.

In particular, the above method can produce a plurality of particleshaving desirable particle size and coefficient of variance. The set ofparticles can include, for example, 100,000 particles, such as 500,000particles, greater than 1 million particles, greater than 10 millionparticles, or even at least 1×10¹⁰. Particles of the plurality ofparticles may be hydrophilic polymeric particles, such as hydrogelparticles. In a particular example, the hydrogel particle can be anacrylamide particle, such as a particle including a crosslinkedhydroxyalkyl acrylamide polymer or a crosslinked copolymer of hydroalkylacrylamide and amine functionalized acrylamide. In another example, theparticle can be a crosslinked copolymer of acrylamide and aminefunctionalized acrylamide.

The plurality of particles can have a desirable particle size, such as aparticle size not greater than 100 μm, not greater than 30 μm, or notgreater than 3 μm. The average particle size is the mean particlediameter. For example, the average particle size may be not greater than2 μm, such as not greater than 1.5 μm, not greater than 1.1 μm, notgreater than 0.8 μm, not greater than 0.6 μm, not greater than 0.5 μm,or even not greater than 0.3 μm. In a particular example, the averageparticle size can be in a range of 0.1 μm to 100 μm, such as a range of0.1 μm to 50 μm or a range of 0.1 μm to 1.1 μm. In some aspects, theabove described method provides technical advantages for production ofparticles having a particle size in a range of 5 μm to 100 μm, such as arange of 20 μm to 100 μm, or a range of 30 μm to 70 μm. In otheraspects, the above described method provides technical advantages forthe production of particles having a particle size of not greater than1.1 μm. When the seed is larger, larger particles can be formed. Thesize of the particles can be adjusted based on the size of the seedparticle. Using the present method, the size of the polymeric particleis less dependent on surfactant selection and concentration than whenother methods are used.

Further, the plurality of particles is monodisperse and may have adesirably low coefficient of variance, such as a coefficient of varianceof not greater than 20%. As above, the coefficient of variance (CV) isdefined as 100 times the standard deviation divided by average, where“average” is the mean particle diameter and standard deviation is thestandard deviation in particle size. The “average” alternatively can beeither the z-average or mode particle diameter. In accordance with usualpractice, CV is calculated on the main mode, i.e., the main peak,thereby excluding minor peaks relating to aggregates. Thus, someparticles below or above mode size may be discounted in the calculationwhich may, for example, be based on about 90% of total particle numberof detectable particles. Such a determination of CV is performable on aCPS disc centrifuge or a coulter counter. For example, the coefficientof variance (CV) of the plurality of particles may be not greater than15%, such as not greater than 10%, not greater than 5%, not greater than4.5%, not greater than 4.0%, not greater than 3.5%, or even not greaterthan 3.0%. Such CV can be accomplished without filtering or other sizeexclusion techniques.

In particular, to keep a low variation of the size of the beads,coalescence of droplets should be avoided during the polymerization.This avoidance is easier to achieve in an oil in water emulsion than ina water in oil emulsion since it is easier to stabilize a system wherewater is the continuous phase. However, hydrophilic monomers do notpreferentially reside in an oil phase.

In a further example, a hydrophilic polymeric particle in water can benot greater than 50 wt % polymer, such as not greater than 30 wt %polymer, not greater than 20 wt % polymer, not greater than 10 wt %polymer, not greater than 5 wt % polymer, or even not greater than 2 wt% polymer.

In an additional example, the polymeric particle can have a porositypermitting diffusion of proteins and enzymes. In an example, thepolymeric particles can have a porosity to permit diffusion of proteinshaving a size of at least 50 kilodaltons, such as at least 100kilodaltons, at least 200 kilodaltons, at least 250 kilodaltons, or evenat least 350 kilodaltons.

In another example, when conjugated, the polymeric particle can includea density of polynucleotides, termed nucleotide density, of at least7×10⁴ per μm³. For example, the nucleotide density can be at least 10⁵per μm³, such as at least 10⁶ per μm³, at least 5×10⁶ per μm³, at least8×10⁶ per μm³, at least 1×10⁷ per μm³, or even at least 3×10⁷ per μm³.In a further example, the nucleotide density can be not greater than10¹⁵ per μm³.

Such polymeric particles can be used in a variety of separationstechniques and analytic techniques. In particular, the polymericparticles may be useful in binding polynucleotides. Such bindingpolynucleotides may be useful in separating polynucleotides fromsolution or can be used for analytic techniques, such as sequencing. Ina particular example illustrated in FIG. 2, such polymeric particles canbe used as a support for polynucleotides during sequencing techniques.For example, such hydrophilic particles can immobilize a polynucleotidefor sequencing using fluorescent sequencing techniques. In anotherexample, the hydrophilic particles can immobilize a plurality of copiesof a polynucleotide for sequencing using ion-sensing techniques.

In general, the polymeric particle can be treated to include abiomolecule, including nucleosides, nucleotides, nucleic acids(oligonucleotides and polynucleotides), polypeptides, saccharides,polysaccharides, lipids, or derivatives or analogs thereof. For example,a polymeric particle can bind or attach to a biomolecule. A terminal endor any internal portion of a biomolecule can bind or attach to apolymeric particle. A polymeric particle can bind or attach to abiomolecule using linking chemistries. A linking chemistry includescovalent or non-covalent bonds, including an ionic bond, hydrogen bond,affinity bond, dipole-dipole bond, van der Waals bond, and hydrophobicbond. A linking chemistry includes affinity between binding partners,for example between: an avidin moiety and a biotin moiety; an antigenicepitope and an antibody or immunologically reactive fragment thereof; anantibody and a hapten; a digoxigen moiety and an anti-digoxigenantibody; a fluorescein moiety and an anti-fluorescein antibody; anoperator and a repressor; a nuclease and a nucleotide; a lectin and apolysaccharide; a steroid and a steroid-binding protein; an activecompound and an active compound receptor; a hormone and a hormonereceptor; an enzyme and a substrate; an immunoglobulin and protein A; oran oligonucleotide or polynucleotide and its corresponding complement.

In an example, the polymeric particle can be utilized in a system with asurface. The system comprises one or more polymeric particles on asurface. A surface can be a solid surface. A surface can include planar,concave, or convex surfaces, or any combination thereof. A surface cancomprise texture or features, including etching, cavitation or bumps. Asurface can lack any texture or features. A surface can include theinner walls of a capillary, channel, groove, well or reservoir. Asurface can be a mesh. A surface can be porous, semi-porous ornon-porous. A surface can be a filter or gel. A surface can include thetop of a pin (e.g., pin arrays). The surface may be made from materialssuch as glass, borosilicate glass, silica, quartz, fused quartz, mica,polyacrylamide, plastic polystyrene, polycarbonate, polymethacrylate(PMA), polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS),silicon, germanium, graphite, ceramics, silicon, semiconductor, highrefractive index dielectrics, crystals, gels, polymers, or films (e.g.,films of gold, silver, aluminum, or diamond). A surface can include asolid substrate having a metal film or metal coat. A surface can beoptically transparent, minimally reflective, minimally absorptive, orexhibit low fluorescence.

A surface can have dimensions similar to microtiter plates having 96,384, 1536, 3456 or 9600 wells. A surface can be about 1-20 cm in any onedimension, about 1-10 cm in any one dimension, about 0.10-1 cm in anyone dimension, or about 0.001 nm-1 cm in any one dimension. A surface(and any texture or features) can be produced by nanofabricationtechnologies.

A plurality of polymeric particles can be arranged in a random orordered array on a surface, or a combination of random and orderedarrays. Ordered arrays include rectilinear and hexagonal patterns. Asurface can include a plurality of sites arranged in a random or orderedarray, or a combination of both. One or more polymeric particles can belocated at one site, some sites or all sites. Some sites can have onepolymeric particle and other sites can have multiple polymericparticles. At least one site can lack a polymeric particle. In an array,at least two polymeric particles can contact each other, or have nocontact between polymeric particles.

As illustrated in FIG. 2, a plurality of polymeric particles 204 can beplaced in a solution along with a plurality of polynucleotides 202. Theplurality of particles 204 can be activated or otherwise prepared tobind with the polynucleotides 202. For example, the particles 204 caninclude an oligonucleotide complementary to a portion of apolynucleotide of the plurality of polynucleotides 202. In anotherexample, the polymeric particles 204 can be modified with targetpolynucleotides 204 using techniques such as biotin-streptavidinbinding.

In a particular embodiment, the hydrophilic particles andpolynucleotides are subjected to polymerase chain reaction (PCR)amplification. For example, dispersed phase droplets 206 or 208 areformed as part of an emulsion and can include a hydrophilic particle ora polynucleotide. In an example, the polynucleotides 202 and thehydrophilic particles 204 are provided in low concentrations and ratiosrelative to each other such that a single polynucleotide 202 is likelyto reside within the same dispersed phase droplets as a singlehydrophilic particle 204. Other droplets, such as a droplet 208, caninclude a single hydrophilic particle and no polynucleotide. Eachdroplet 206 or 208 can include enzymes, nucleotides, salts or othercomponents sufficient to facilitate duplication of the polynucleotide.

In a particular embodiment, an enzyme such as a polymerase is present,bound to, or is in close proximity to the hydrophilic particle orhydrogel particle of the dispersed phase droplet. In an example, apolymerase is present in the dispersed phase droplet to facilitateduplication of the polynucleotide. A variety of nucleic acid polymerasemay be used in the methods described herein. In an exemplary embodiment,the polymerase can include an enzyme, fragment or subunit thereof, whichcan catalyze duplication of the polynucleotide. In another embodiment,the polymerase can be a naturally-occurring polymerase, recombinantpolymerase, mutant polymerase, variant polymerase, fusion or otherwiseengineered polymerase, chemically modified polymerase, syntheticmolecules, or analog, derivative or fragment thereof.

In an embodiment, the polymerase can be any Family A DNA polymerase(also known as pol I family) or any Family B DNA polymerase. Inembodiments, the DNA polymerase can be a recombinant form capable ofduplicating polynucleotides with superior accuracy and yield as comparedto a non-recombinant DNA polymerase. For example, the polymerase caninclude a high-fidelity polymerase or thermostable polymerase. Inembodiments, conditions for duplication of polynucleotides can include‘Hot Start’ conditions, for example Hot Start polymerases, such asAmplitaq Gold® DNA polymerase (Applied Biosciences) or KOD Hot Start DNApolymerase (EMD Biosciences). Typically, a ‘Hot Start’ polymeraseincludes a thermostable polymerase and one or more antibodies thatinhibit the DNA polymerase and 3′-5′ exonuclease activities at ambienttemperature.

In embodiments, the polymerase can be an enzyme such as Taq polymerase(from Thermus aquaticus), Tfi polymerase (from Thermus filiformis), Bstpolymerase (from Bacillus stearothermophilus), Pfu polymerase (fromPyrococcus furiosus), Tth polymerase (from Thermus thermophilus), Powpolymerase (from Pyrococcus woesei), Tli polymerase (from Thermococcuslitoralis), Ultima polymerase (from Thermotoga maritima), KOD polymerase(from Thermococcus kodakaraensis), Pol I and II polymerases (fromPyrococcus abyssi) and Pab (from Pyrococcus abyssi).

In embodiments, the polymerase can be a recombinant form of Thermococcuskodakaraensis. In embodiments, the polymerase can be a KOD or KOD-likeDNA polymerase such as KOD polymerase (EMD Biosciences), KOD “Hot Start”polymerase (EMD Biosciences), KOD Xtreme Hot Start DNA Polymerase (EMDBiosciences), KOD XL DNA polymerase (EMD Biosciences), Platinum® Taq DNAPolymerase (Invitrogen), Platinum® Taq DNA Polymerase High Fidelity(Invitrogen), Platinum® Pfx (Invitrogen), Accuprime™ Pfx (Invitrogen),Accuprime™ Taq DNA Polymerase High Fidelity (Invitrogen) or AmplitaqGold® DNA Polymerase (Applied Biosystems). In embodiments, thepolymerase can be a DNA polymerase containing analogous mutations tothose polymerases discussed herein.

In embodiments, duplication of the polynucleotide can include modulatingthe duplication conditions. Modulating can optionally include:increasing or decreasing the polymerase concentration; increasing ordecreasing the nucleotide concentration; increasing or decreasing acation concentration; increasing or decreasing a reaction temperature,time or pH, or the like. The modulating can include increasing ordecreasing the rate of the reaction, increasing or decreasing the yieldof product of the reaction, or the like. In embodiments, duplication canbe performed in the presence of appropriate buffers or nucleotides(including nucleotide analogs or biotinylated nucleotides).

In particular, the polynucleotide to be amplified can be captured by thepolymeric particle. Exemplary methods for capturing nucleic acid caninclude: hybridizing a polynucleotide to an oligonucleotide that isattached to a polymeric particle. In embodiments, methods for capturingnucleic acids comprise: (a) providing a polymeric particle attached to asingle-stranded oligonucleotide (e.g., a capture oligonucleotide); (b)providing a single-stranded polynucleotide; and (c) hybridizing thesingle-stranded oligonucleotide to the single-stranded polynucleotides,thereby capturing the single-stranded polynucleotide to the polymericparticle. In embodiments, each of the polymeric particles can beattached with a plurality of single-stranded oligonucleotides (e.g.,capture oligonucleotides). In embodiments, step (c) can be conductedwith a plurality of single-stranded polynucleotides. In embodiments, atleast a portion of the single-stranded oligonucleotide comprises anucleotide sequence that is complementary (or partially complementary)to at least a portion of the single-stranded polynucleotide.

In an example, the method further includes amplifying the polynucleotideinto a plurality of polynucleotides and attaching at least a portion ofthe plurality of polynucleotides to the hydrophilic particle, therebygenerating a hydrophilic particle including a plurality of attachedpolynucleotides. Alternatively, the method can further includeamplifying the polynucleotide into a plurality of complementarypolynucleotides by extending the oligonucleotide, thereby generating ahydrogel particle including a plurality of attached polynucleotides.

In embodiments, methods for nucleotide incorporation comprise:conducting a nucleotide polymerization reaction on a polynucleotide thatis hybridized to an oligonucleotide that is attached to a polymericparticle. In embodiments, methods for nucleotide incorporation comprise:(a) providing a polymeric particle attached to a single-strandedoligonucleotide (e.g., a primer oligonucleotide); (b) providing asingle-stranded template polynucleotide; (c) hybridizing thesingle-stranded oligonucleotide to the single-stranded templatepolynucleotide; and (d) contacting the single-stranded templatepolynucleotide with a polymerase and at least one nucleotide underconditions suitable for the polymerase to catalyze polymerization of atleast one nucleotide onto the single-stranded oligonucleotide, therebyconducting nucleotide incorporation. In embodiments, each of thepolymeric particles can be attached with a plurality of single-strandedoligonucleotides (e.g., capture oligonucleotides). In embodiments, steps(b), (c) or (d) can be conducted with a plurality of single-strandedpolynucleotides. In embodiments, at least a portion of thesingle-stranded oligonucleotide comprises a nucleotide sequence that iscomplementary (or partially complementary) to at least a portion of thesingle-stranded polynucleotide. In embodiments, a system comprises asingle-stranded polynucleotide hybridized to a single-strandedoligonucleotide which is attached to a polymeric particle, wherein atleast one nucleotide is polymerized onto the end of the single-strandedoligonucleotide.

In embodiments, methods for primer extension comprise: conducting aprimer extension reaction on a polynucleotide that is hybridized to anoligonucleotide that is attached to a polymeric particle. Inembodiments, methods for nucleic acid primer extension comprise: (a)providing a polymeric particle attached to a single-strandedoligonucleotide (e.g., a primer oligonucleotide); (b) providing asingle-stranded template polynucleotide; (c) hybridizing thesingle-stranded oligonucleotide to the single-stranded templatepolynucleotide; and (d) contacting the single-stranded templatepolynucleotide with a polymerase and at least one nucleotide underconditions suitable for the polymerase to catalyze polymerization of atleast one nucleotide onto the single-stranded oligonucleotide, therebyextending the primer. In embodiments, each of the polymeric particlescan be attached with a plurality of single-stranded oligonucleotides(e.g., capture oligonucleotides). In embodiments, step (b), (c) or (d)can be conducted with a plurality of single-stranded polynucleotides. Inembodiments, at least a portion of the single-stranded oligonucleotidecomprises a nucleotide sequence that is complementary (or partiallycomplementary) to at least a portion of the single-strandedpolynucleotide. In embodiments, a system comprises a single-strandedpolynucleotide hybridized to a single-stranded oligonucleotide which isattached to a polymeric particle, wherein the single-strandedoligonucleotide is extended with one or more nucleotides.

In embodiments, methods for nucleic acid amplification comprise:conducting a primer extension reaction on a polynucleotide that ishybridized to an oligonucleotide which is attached to a polymericparticle. In embodiments, methods for nucleic acid amplificationcomprise: (a) providing a polymeric particle attached to asingle-stranded oligonucleotide (e.g., a primer oligonucleotide); (b)providing a single-stranded template polynucleotide; (c) hybridizing thesingle-stranded oligonucleotide to the single-stranded templatepolynucleotide; (d) contacting the single-stranded templatepolynucleotide with a polymerase and at least one nucleotide underconditions suitable for the polymerase to catalyze polymerization of atleast one nucleotide onto the single-stranded oligonucleotide so as togenerate an extended single-stranded oligonucleotide. In embodiments,the method further comprises: (e) removing (e.g., denaturing) thesingle-stranded template polynucleotide from the extendedsingle-stranded oligonucleotide so that the single-strandedoligonucleotide remains attached to the polymeric particle; (f)hybridizing the remaining single-stranded oligonucleotide to a secondsingle-stranded template polynucleotide; and (g) contacting the secondsingle-stranded template polynucleotide with a second polymerase and asecond at least one nucleotide, under conditions suitable for the secondpolymerase to catalyze polymerization of the second at least onenucleotide onto the single-stranded oligonucleotide so as to generate asubsequent extended single-stranded oligonucleotide. In embodiments,steps (e), (f) and (g) can be repeated at least once. In embodiments,the polymerase and the second polymerase comprise a thermostablepolymerase. In embodiments, the conditions suitable for nucleotidepolymerization include conducting the nucleotide polymerization steps(e.g., steps (d) or (g)) at an elevated temperature. In embodiments, theconditions suitable for nucleotide polymerization include conducting thenucleotide polymerization step (e.g., steps (d) or (g)) at alternatingtemperatures (e.g., an elevated temperature and a relatively lowertemperature). In embodiments, the alternating temperature ranges from60-95° C. In embodiments, the temperature cycles can be about 10 secondsto about 5 minutes, or about 10 minutes, or about 15 minutes, or longer.In embodiments, methods for nucleic acid amplification can generate oneor more polymeric particles each attached to a plurality of templatepolynucleotides comprising sequences that are complementary to thesingle-stranded template polynucleotide or to the second single-strandedtemplate polynucleotide. In embodiments, each of the polymeric particlescan be attached with a plurality of single-stranded oligonucleotides(e.g., capture oligonucleotides). In embodiments, step (b), (c), (d),(e), (f) or (g) can be conducted with a plurality of single-strandedpolynucleotides. In embodiments, at least a portion of thesingle-stranded oligonucleotide comprises a nucleotide sequence that iscomplementary (or partially complementary) to at least a portion of thesingle-stranded polynucleotide. In embodiments, methods for nucleic acidamplification (as described above) can be conducted in an aqueous phasesolution in an oil phase (e.g., dispersed phase droplet).

Following PCR, particles are formed, such as particle 210, which caninclude the hydrophilic particle 212 and a plurality of copies 214 ofthe polynucleotide. While the polynucleotides 214 are illustrated asbeing on a surface of the particle 210, the polynucleotides can extendwithin the particle 210. Hydrogel and hydrophilic particles having a lowconcentration of polymer relative to water can include polynucleotidesegments on the interior of and throughout the particle 210 orpolynucleotides can reside in pores and other openings. In particular,the particle 210 can permit diffusion of enzymes, nucleotides, primersand reaction products used to monitor the reaction. A high number ofpolynucleotides per particle produces a better signal.

In embodiments, polymeric particles from an emulsion-breaking procedurecan be collected and washed in preparation for sequencing. Collectioncan be conducted by contacting biotin moieties (e.g., linked toamplified polynucleotide templates which are attached to the polymericparticles) with avidin moieties, and separation away from polymericparticles lacking biotinylated templates. Collected polymeric particlesthat carry double-stranded template polynucleotides can be denatured toyield single-stranded template polynucleotides for sequencing.Denaturation steps can include treatment with base (e.g., NaOH),formamide, or pyrrolidone.

In an exemplary embodiment, the particle 210 can be utilized in asequencing device. For example, a sequencing device 216 can include anarray of wells 218. A particle 210 can be placed within a well 218.

In an example, a primer can be added to the wells 218 or the particle210 can be pre-exposed to the primer prior to placement in the well 218.In particular, the particle 210 can include bound primer. The primer andpolynucleotide form a nucleic acid duplex including the polynucleotide(e.g., a template nucleic acid) hybridized to the primer. The nucleicacid duplex is an at least partially double-stranded polynucleotide.Enzymes and nucleotides can be provided to the well 218 to facilitatedetectable reactions, such as nucleotide incorporation.

Sequencing can be performed by detecting nucleotide addition. Nucleotideaddition can be detected using methods such as fluorescent emissionmethods or ion detection methods. For example, a set of fluorescentlylabeled nucleotides can be provided to the system 216 and can migrate tothe well 218. Excitation energy can be also provided to the well 218.When a nucleotide is captured by a polymerase and added to the end of anextending primer, a label of the nucleotide can fluoresce, indicatingwhich type of nucleotide is added.

In an alternative example, solutions including a single type ofnucleotide can be fed sequentially. In response to nucleotide addition,the pH within the local environment of the well 218 can change. Such achange in pH can be detected by ion sensitive field effect transistors(ISFET). As such, a change in pH can be used to generate a signalindicating the order of nucleotides complementary to the polynucleotideof the particle 210.

In particular, a sequencing system can include a well, or a plurality ofwells, disposed over a sensor pad of an ionic sensor, such as a fieldeffect transistor (FET). In embodiments, a system includes one or morepolymeric particles loaded into a well which is disposed over a sensorpad of an ionic sensor (e.g., FET), or one or more polymeric particlesloaded into a plurality of wells which are disposed over sensor pads ofionic sensors (e.g., FET). In embodiments, an FET can be a chemFET or anISFET. A “chemFET” or chemical field-effect transistor, includes a typeof field effect transistor that acts as a chemical sensor. The chemFEThas the structural analog of a MOSFET transistor, where the charge onthe gate electrode is applied by a chemical process. An “ISFET” orion-sensitive field-effect transistor, can be used for measuring ionconcentrations in solution; when the ion concentration (such as H+)changes, the current through the transistor changes accordingly.

In embodiments, the FET may be a FET array. As used herein, an “array”is a planar arrangement of elements such as sensors or wells. The arraymay be one or two dimensional. A one dimensional array can be an arrayhaving one column (or row) of elements in the first dimension and aplurality of columns (or rows) in the second dimension. The number ofcolumns (or rows) in the first and second dimensions may or may not bethe same. The FET or array can comprise 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷ ormore FETs.

In embodiments, one or more microfluidic structures can be fabricatedabove the FET sensor array to provide for containment or confinement ofa biological or chemical reaction. For example, in one implementation,the microfluidic structure(s) can be configured as one or more wells (ormicrowells, or reaction chambers, or reaction wells, as the terms areused interchangeably herein) disposed above one or more sensors of thearray, such that the one or more sensors over which a given well isdisposed detect and measure analyte presence, level, or concentration inthe given well. In embodiments, there can be a 1:1 correspondence of FETsensors and reaction wells.

Returning to FIG. 2, in another example, a well 218 of the array ofwells can be operatively connected to measuring devices. For example,for fluorescent emission methods, a well 218 can be operatively coupledto a light detection device. In the case of ionic detection, the lowersurface of the well 218 may be disposed over a sensor pad of an ionicsensor, such as a field effect transistor.

One exemplary system involving sequencing via detection of ionicbyproducts of nucleotide incorporation is the Ion Torrent PGM™ sequencer(Life Technologies), which is an ion-based sequencing system thatsequences nucleic acid templates by detecting hydrogen ions produced asa byproduct of nucleotide incorporation. Typically, hydrogen ions arereleased as byproducts of nucleotide incorporations occurring duringtemplate-dependent nucleic acid synthesis by a polymerase. The IonTorrent PGM™ sequencer detects the nucleotide incorporations bydetecting the hydrogen ion byproducts of the nucleotide incorporations.The Ion Torrent PGM™ sequencer can include a plurality of templatepolynucleotides to be sequenced, each template disposed within arespective sequencing reaction well in an array. The wells of the arraycan each be coupled to at least one ion sensor that can detect therelease of H+ ions or changes in solution pH produced as a byproduct ofnucleotide incorporation. The ion sensor comprises a field effecttransistor (FET) coupled to an ion-sensitive detection layer that cansense the presence of H+ ions or changes in solution pH. The ion sensorcan provide output signals indicative of nucleotide incorporation whichcan be represented as voltage changes whose magnitude correlates withthe H+ ion concentration in a respective well or reaction chamber.Different nucleotide types can be flowed serially into the reactionchamber, and can be incorporated by the polymerase into an extendingprimer (or polymerization site) in an order determined by the sequenceof the template. Each nucleotide incorporation can be accompanied by therelease of H+ ions in the reaction well, along with a concomitant changein the localized pH. The release of H+ ions can be registered by the FETof the sensor, which produces signals indicating the occurrence of thenucleotide incorporation. Nucleotides that are not incorporated during aparticular nucleotide flow may not produce signals. The amplitude of thesignals from the FET can also be correlated with the number ofnucleotides of a particular type incorporated into the extending nucleicacid molecule thereby permitting homopolymer regions to be resolved.Thus, during a run of the sequencer multiple nucleotide flows into thereaction chamber along with incorporation monitoring across amultiplicity of wells or reaction chambers can permit the instrument toresolve the sequence of many nucleic acid templates simultaneously.Further details regarding the compositions, design and operation of theIon Torrent PGM™ sequencer can be found, for example, in U.S. patentapplication Ser. No. 12/002,781, now published as U.S. PatentPublication No. 2009/0026082 and issued U.S. Pat. No. 8,262,900; U.S.patent application Ser. No. 12/474,897, now published as U.S. PatentPublication No. 2010/0137143; and U.S. patent application Ser. No.12/492,844, now published as U.S. Patent Publication No. 2010/0282617,all of which applications are incorporated by reference herein in theirentireties.

Embodiments of the polymeric particles exhibit technical advantages whenused in sequencing techniques, particularly ion-based sequencingtechniques. In particular, embodiments of the polymeric particles arenon-buffering or enhance read lengths or accuracy.

In a further example, the polymeric particles can exhibit greateruniformity and lower CV without filtering than particles made throughother methods. For example, the above methods can directly form thepolymer particles without applying any kind of selection process such asfiltering or using a centrifuge. In particular, emulsion polymerizationcan be used to produce particles suitable for seed particles. Typicallyseed particles are non-crosslinked to be able to adsorb the promotermolecule.

Normally styrene is used to produce seed particles with a CV of lessthan 5%, and reports of other monomers is limited. Unexpectedly andadvantageously, tBDMS HEAM can be used to produce seeds with a CV of 2%using a method, such as the emulsion polymerization method, describedabove.

Further, embodiments of the present method provide for size controlbased on the size of the seed particle. Additionally, embodiments ofparticles made by such methods provide an increase in conjugation, suchas a 60% to 80% increase in conjugation, over other methods.

For example, when measured on an Ion Torrent 314 PGM, embodiments ofconjugated polymeric particles exhibit Q17 mean read lengths of at least200 bp, such as at least 250 bp, at least 300 bp, at least 350 bp, oreven at least 400 bp. In particular, conjugated polymeric particles canexhibit Q17 mean read lengths of at least 500 bp. In another example,populations of conjugated polymeric particles exhibit a 200Q17 run of atleast 200K, such as at least 300K, at least 350K, or even at least 400K.

EXAMPLES Example 1

A silyl protected acrylamide monomer,(N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide), is formed from ahydroxyalkylacrylamide monomer and a halogenated silyl group.

t-Butyldimethylsilyl chloride (66.11 g, 439 mmol) is added in threeportions at 30 min intervals to a solution of hydroxyethylacrylamide(50.01 g, 434 mmol) and imidazole (73.94 g, 1086 mmol) in DMF (132 g) at0° C. under an inert atmosphere (Ar). Extra DMF (18.90 g) is added 15minutes after the last addition. The reaction mixture is allowed togradually reach room temperature and is stirred for approximately 24hours. The reaction mixture is quenched with water (101 g) and stirredfor one hour. The mixture is then extracted with diethyl ether andwashed with water and brine. The organic phase is dried (MgSO₄ overnight. Evaporation gives 93.59 g product. 4-methoxyphenol (MEHQ) (9 mg,100 ppm) is added towards the end of the evaporation. The product isstored at −20° C.

Example 2

A silyl protected acrylamide monomer,(N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide) (tBDMS-HEAM), ispolymerized in a dispersed phase and is deprotected to form a hydrogelparticle.

An initiator emulsion is formed by mixing 1.14 g sodium dodecyl sulfate(SDS), 190 g water, 9.50 g acetone, and 19.0 g dioctanoylperoxide withan ultraturax type Ystral™ X10/25 homogeniser (“ultraturax”) for 2minutes and is homogenized with a pressure homogenizer for 7 minutes.

In a 0.5 L flask, 202.42 g of a 0.53 micron monodisperse polystyreneseed dispersion with 7.2 w % solids content is mixed with 115.65 g ofthe initiator emulsion. The mixture is stirred at 26° C. for 20 hoursgiving a promoted seed solution.

A PVP solution is formed from 16.02 g polyvinylpyrrolidone (PVP) K-30.K-30 is slowly added to 959 g water and stirred for 30 minutes, followedby addition of 1.08 g SDS.

A buffer solution is prepared from 47.99 g sodium hydrogen carbonateadded to 912 g water.

A monomer emulsion is prepared from 19.63 g toluene, 11.49 g tBDMS-HEAM,0.77 g ethylene glycol dimethacrylate (EDMA), 187.42 g water and 186.4 gPVP solution mixed by ultraturax for 2 minutes, and further homogenizedfor 5 minutes.

In a 0.5 L reactor, 29.45 g of the promoted seed particles and 189.93 gof the monomer emulsion is added, followed by 26.65 g buffer solution.The mixture is stirred for 2 hours at 25° C. and then 53.33 g water isadded. The mixture is heated to 60° C. After 1 hour at 60° C., thetemperature is raised to 70° C. and maintained at 70° C. for 5 hours.

The reaction mixture is transferred to a 1 liter centrifugation flaskand centrifuged in a Sorvall RC3CPlus centrifuge for 60 minutes at 4500RPM. The creamy flotation product is transferred to a new 1 liter flaskand is centrifuge twice in tetrahydrofuran (THF).

The THF swollen gel sediment is mixed with glacial acetic acid and waterto a weight ratio of 1:3:1 and shaken at room temperature over night.The gel is worked by removing the supernatant after centrifugation andadding a mixture of THF and water in a ratio THF:water 1:1, two timesand water once, followed by three times with dimethylformamide (DMF) andthree times with dry DMF.

Example 3

A silyl protected acrylamide monomer,(N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide) (tBDMS-HEAM), ispolymerized in a dispersed phase formed from polystyrene particles andis deprotected to form a hydrogel particle.

A carbonate buffer solution is prepared from 42.1 g sodium hydrogencarbonate added to 800 g water to give a 0.5 M buffer solution. The pHof the buffer is adjusted to 10 with 0.5 M sodium hydroxide.

An initiator emulsion is prepared from 0.96 g SDS, 160 g water, 8.00 gacetone, and 16.0 g dioctanoylperoxide. The pH is adjusted to 9 with 0.5M carbonate buffer solution. The mixture is homogenized with anultraturax type Ystral™ X10/25 homogeniser (“ultraturax”) for 2 minutesand homogenized with a pressure homogenizer for 6 minutes.

In a 0.5 L flask, 31.2 g of 0.31 micron monodisperse polystyrene seeddispersion with 15.91 w % solids content is mixed with 103.1 g of theinitiator emulsion. The mixture is stirred at 26° C. for 20 hours,giving a promoted seed dispersion.

A PVA solution is prepared by slowly adding 80 g of 87-89% hydrolyzedpolyvinylalcohol (PVA) to 2000 g water, stirring and heating to 80° C.for 1 hour and cooling. An amount of 91 g of the PVA solution is mixedwith 867 g water, 0.74 g SDS and 4.24 g 0.5 M sodium carbonate buffer.

The monomer emulsion is prepared from 29.2 g toluene, 7.71 g tBDMS-HEAM,0.76 g EDMA, and 249 g PVA solution mixed by ultraturax for 2 minutes,and further homogenized for 5 minutes.

In a 0.5 L reactor, 13.3 g of the promoted seed particles and 287 g ofthe monomer emulsion is added. The mixture is stirred for 1 hour at 25°C. and is heated to 50° C. After 1 hour at 50° C., the temperature israised to 70° C. After two hours, 2.8 ml of 0.5 M buffer solution isadded, and the temperature is maintained for one more hour.

The reaction mixture is transferred to a 1 liter centrifugation flaskand centrifuged in a Sorvall RC3CPlus centrifuge for 60 minutes at 4500RPM. The creamy flotation product is transferred to a new 1 liter flaskand is centrifuged twice in THF.

To 102 g of the THF swollen gel sediment, glacial acetic acid and wateris added in a weight ratio of 1:3:1 and the dispersion is shaken at roomtemperature over night. The gel is worked by removing the supernatantafter centrifugation and adding THF and water in a ratio of THF:water1:1 twice, followed by three centrifugations with DMF and threecentrifugation with dry DMF.

The solids content of the product is determined to be 0.48 g and thediameter of the bead in water was determined to be 1.6 micron bymicroscopy.

Example 4

Hydrogel particles formed in accordance with Example 3 are activatedusing tresyl chloride.

In particular, 26 g of the DMF dispersion containing 3.08% hydroxyl gelfrom Example 3 above is washed three times with 30 ml anhydrous DMF bycentrifugation and removing the supernatant. After the lastcentrifugation, the volume of the gel in DMF is adjusted to 26 mL andthe tube is shortly flushed with argon. An amount of 0.241 ml ofanhydrous pyridine is added, followed by 0.318 g tresyl chloride. Thetube is shaken over night. The dispersion is centrifuged with 30 ml icecold anhydrous DMF four times, removing the supernatant after eachcentrifugation, and is centrifuged two times with ice cold anhydrousN-methyl-2-pyrrolidone (NMP). The particles are re-suspend in 50 mL (2%dry) of anhydrous NMP.

Example 5

Hydrogel particles formed in accordance with Example 3 are activatedusing fosyl chloride.

An amount of 26 g of DMF dispersion containing 3.08% hydroxyl gel fromExample 3 is washed three times with 30 ml anhydrous DMF bycentrifugation and removing the supernatant. After the lastcentrifugation, the volume of the gel in DMF is adjusted to 26 mL andthe tube is shortly flushed with argon. An amount of 0.080 ml anhydrouspyridine is added, followed by 0.113 g fosyl chloride. The tube isshaken over night and centrifuged, removing the supernatant and adding30 ml of ice cold anhydrous DMF four times and ice cold anhydrous NMPtwice. The particles are re-suspend in 50 mL (2% dry) of anhydrous NMP.

Example 6

Hydrogel particles formed in accordance with Example 3 are activatedusing mesyl chloride.

An amount of 16 g of DMF containing 3.08% hydroxyl gel from Example 3 iswashed three times with 30 ml anhydrous DMF by centrifugation andremoving the supernatant. After the last centrifugation, the volume ofthe gel in DMF is adjusted to 16 mL and the tube is shortly flushed withargon. An amount of 0.049 ml anhydrous pyridine is added, followed by0.121 g mesyl chloride. The tube is shaken over night and centrifuged,removing the supernatant and adding 30 ml of ice cold anhydrous DMF fourtimes and ice cold anhydrous NMP twice. The particles are re-suspend in50 mL (2% dry) of anhydrous NMP.

Example 7

A silyl protected acrylamide monomer,(N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide) (tBDMS-HEAM), ispolymerized with divinyl benzene crosslinker in a dispersed phase formedfrom polystyrene particles and is deprotected to form a hydrogelparticle.

An initiator emulsion is prepared from 1.26 g SDS, 210 g water, 10.5 gacetone and 21.0 g dioctanoylperoxide mixed with an ultraturax typeYstral™ X10/25 homogeniser (“ultraturax”) for 2 minutes and homogenizedwith a pressure homogenizer for 7 minutes.

In a 0.5 L flask, 40.7 g of 0.31 micron monodisperse polystyrene seeddispersion with 15.9 w % solids content is mixed with 142.2 g of theinitiator emulsion. The mixture is stirred at 26° C. for 48 hours,giving a promoted seed dispersion.

A PVA solution is prepared by slowly adding 80 g polyvinylalcohol (PVA)to 2000 g water and stirring and heating to 80° C. for 1 hour. The PVAsolution is subsequently cooled.

To 208 g of the concentrated PVA solution is added 1806 g water, 1.76 gSDS and 7.68 g borax, forming a PVA borax solution.

Amounts of 31.08 g toluene, 9.79 g tBDMS HEAM, 0.37 g 80% divinylbenzene(DVB) (comprising 0.296 g DVB and 0.074 g ethylvinylbenzene), 273.7 gPVA borax solution are mixed by ultraturax for 2 minutes, and furtherhomogenized for 5 minutes to form a monomer emulsion.

In a 0.5 L reactor, 10.1 g of the promoted seed particles is mixed with289.9 g of the monomer emulsion. The mixture is stirred for 1 hour at30° C., 1 hour at 50° C., and 2 hours at 75° C.

The reaction mixture is transferred to a 1 liter centrifugation flaskand centrifuged in a Sorvall RC3CPlus centrifuge for 80 minutes at 4500RPM. The creamy flotation product is collected and centrifuged six timesin THF.

To 160 g of the THF swollen gel sediment is added glacial acetic acidand water in a weight ratio of 1:3:1. The dispersion is shaken at roomtemperature over night. The gel is worked by removing the supernatantafter centrifugation and adding THF and water in a ratio of THF:water1:1 two times and water once, followed by three times with DMF. Thesolids content of the DMF is then determined to be 1.01 g. The beaddispersion was transferred to water and inspected by microscopy. Thebeads were monosized and the bead diameter is 1.9 micron.

Example 8

A silyl protected acrylamide monomer,(N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide) (tBDMS-HEAM), ispolymerized with a protected crosslinker in a dispersed phase formedfrom polystyrene particles and is deprotected to form a hydrogelparticle.

A concentrated PVA solution is prepared from 80 g polyvinylalcohol (PVA)slowly added to 2000 g water. The solution is stirred and heated to 80°C. for 1 hour and is cooled.

To 160.5 g of the concentrated PVA solution, 1425 g water, 1.56 g SDSand 6.06 g borax are added. The pH of the solution is adjusted to 8.2with 2M HCl.

Amounts of 11.8 g toluene, 11.80 g 3-phenylpropanol, 0.15 g2,2′-azobis-(2-methylbutyronitrile) (AMBN), 7.78 g tBDMS HEAM, 1.44 gN,N′-(N-(2-((triethylsilyl)oxy)propane-1,3-diyl)diacrylamide (TES-PBAM)(82% purity) and 291.6 g PVA borax solution are mixed by ultraturax for2 minutes, and further homogenized for 5 minutes to form a monomeremulsion.

In a 0.5 L reactor, 5.96 g of a water dispersion of polystyrene seedparticles (seed diameter 0.385 μm, 8.08 weight % solids) is mixed with294.5 g of the monomer emulsion. Argon gas (10-20 ml/min) is bubbledthrough the mixture, while stirring and heating 1 hours at 30° C. and 1hour at 50° C. The argon flow is stopped, and heating and stirringcontinued for 3 hours at 80° C.

The reaction mixture is transferred to a 1 liter centrifugation flaskand centrifuged in a Sorvall RC3CPlus centrifuge for 50 minutes at 4500RPM. The creamy flotation product is collected and centrifuged fourtimes in THF.

To 209 g of the THF swollen gel sediment is added glacial acetic acid209 g and water 105 g. The mixture is shaken at room temperatureovernight. The gel is worked up by removing the supernatant aftercentrifugation and adding THF and water in a ratio of THF:water 1:1 twotimes and water once, followed by three times with DMF.

The solids content of the dispersion is determined to be 1.97 g. Thediameter of a water swollen gel can be measured in a microscope withphase contrast equipment to 1.9 μm. The CV is not greater than 5.0%.

Example 9

A silyl protected acrylamide monomer,(N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide) (tBDMS-HEAM), ispolymerized with aN,N′-(ethane-1,2-diyl)bis(N-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide(tBDMS EBHEAM) crosslinker in a dispersed phase formed from polystyreneparticles and is deprotected to form a hydrogel particle.

A concentrated PVA solution is formed from 80 g polyvinylalcohol (PVA)slowly added to 2000 g water, followed by stirring and heating to 80° C.for 1 hour and cooling.

To 88 g of the concentrated PVA solution, 785 g water, 0.88 g SDS, and3.33 g borax are added to from a PVA borax solution.

A monomer emulsion is formed from 7.82 g toluene, 0.040 g2,2′-azobis-(2-methylbutyronitrile) (AMBN), 2.06 g tBDMS HEAM, 0.51 gtBDMS-EBHEAM (95 purity) and 92.9 g PVA borax solution mixed byultraturax for 2 minutes, and further homogenized for 5 minutes.

In a 0.5 L reactor, 1.65 g of a water dispersion of seed particles (seeddiameter 0.319 μm, 8.07 weight % solids) is mixed with 88.34 g of themonomer emulsion. Argon gas (10-20 ml/min) is bubbled through themixture while stirring and heating 1 hour at 30° C. and 2 hours at 40°C. The argon flow is stopped, and heating and stirring continued for 3hours at 80° C.

The reaction mixture is transferred to a 1 liter centrifugation flaskand centrifuged in a Sorvall RC3CPlus centrifuge for 50 minutes at 4500RPM. The creamy flotation product is collected and is centrifuged twicein THF.

To 83.9 g of the THF swollen gel sediment, the same weight of glacialacetic acid and half the weight of water is added. The mixture is shakenat room temperature overnight. The gel is worked by removing thesupernatant after centrifugation and adding THF and water in a ratio ofTHF:water 1:1 two times and water once, followed by three times withDMF.

The solids content of the dispersion is determined to be 1.63 g. Thediameter of a water swollen gel can be measured in a microscope withphase contrast equipment and is on average 1.6 μm. The CV is not greaterthan 5.0%.

Example 10

A silyl protected acrylamide monomer,(N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide) (tBDMS-HEAM), ispolymerized with a divinyl benzene (DVB) crosslinker in a dispersedphase formed from polystyrene particles and is deprotected to form ahydrogel particle.

An initiator emulsion is prepared from 1.2 g SDS, 200 g water, 10 gacetone and 20.0 g dioctanoylperoxide mixed with an ultraturax typeYstral™ X10/25 homogeniser (“ultraturax”) for 2 minutes and homogenizedwith a pressure homogenizer for 7 minutes.

In a 0.5 L flask, 31.64 g of 0.13 micron monodisperse polystyrene seeddispersion with 4.55 w % solids content is mixed with 15.84 g of theinitiator emulsion. The mixture is stirred at 26° C. for 6 days, givinga promoted seed dispersion.

A borax solution is prepared with 1922 g water mixed with 5.92 g SDS and7.33 g borax.

A monomer emulsion is prepared from 172.4 g toluene, 54.76 g tBDMS HEAM,2.75 g 80% divinylbenzene (DVB) (comprising 2.2 g DVB and 0.55 gethylvinylbenzene) and 1468 g of the borax solution mixed by ultraturaxand further homogenized for 17 minutes.

In a 0.5 L reactor, 22.6 g of the promoted seed particles is mixed with277.6 g of the monomer emulsion. The mixture is stirred for 1 hour at30° C., 1 hour at 40° C. and 2 hours at 75° C.

The reaction mixture is transferred to a 1 liter centrifugation flaskand centrifuged in a Sorvall RC3CPlus centrifuge for 90 minutes at 4500RPM. The creamy flotation product is collected and is centrifuged fourtimes in THF.

To 73.3 g of THF swollen gel sediment, an equal weight of glacial aceticacid and 36.7 g water are added, and the dispersion is shaken at roomtemperature over night. The gel is worked by removing the supernatantafter centrifugation and adding THF and water in ratios THF:water 7:3and THF:water 6:4; followed by three centrifugations with DMF.

The solids content of the dispersion is determined to be 1.50 g.

Example 11

A silyl protected acrylamide monomer,(N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide) (tBDMS-HEAM), ispolymerized with a DVB crosslinker in a dispersed phase formed frompolystyrene particles and is deprotected to form a hydrophilic particle.

A concentrated PVA solution is prepared from 80 g polyvinylalcohol (PVA)slowly added to 2000 g water. The dispersion is stirred and is heated to80° C. for 1 hour and cooled.

To 208 g of the concentrated PVA solution, 1814 g water, 2.08 g SDS and7.71 g borax is added to form a PVA borax solution.

A monomer emulsion is prepared from 22.34 g toluene, 0.164 g2,2′-azobis-(2-methylbutyronitrile) (AMBN), 16.58 g tBDMS HEAM, 3.46 g80% divinylbenzene (comprising 2.77 g DVB and 0.69 g ethylvinylbenzene)and 275 g PVA borax solution mixed by ultraturax for 2 minutes, andfurther homogenized for 5 minutes.

In a 0.5 L reactor, 8.46 g of a water dispersion of seed particles (seeddiameter 0.550 μm, 7.20 weight % solids) is mixed with 292.15 g of themonomer emulsion. The mixture is stirred and heated for 1 hour at 30°C., 1 hour at 50° C. and 2 hours at 75° C.

The reaction mixture is transferred to a 1 liter centrifugation flaskand centrifuged in a Sorvall RC3CPlus centrifuge for 50 minutes at 4500RPM. The creamy flotation product is collected and is centrifuged twicein THF.

To 83.9 g of the THF swollen gel sediment, the same weight of glacialacetic acid and half the weight of water are added. The mixture isshaken at room temperature overnight. The gel is worked by removing thesupernatant after centrifugation and adding THF and water in a ratio ofTHF:water 1:1 two times and water once, followed by three times withDMF.

The solids content of the DMF is 7.5 g. The diameter of a water swollengel can be measured in a microscope with phase contrast equipment and isdetermined to be 1.8 μm on average. The CV is not greater than 5.0%.

Example 12

A silyl protected acrylamide monomer,(N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide) (tBDMS-HEAM), ispolymerized with a tBDMS-EBHEAM crosslinker in a dispersed phase formedfrom polystyrene particles and is deprotected to form a hydrogelparticle.

A concentrated PVA solution is formed from 80 g polyvinylalcohol (PVA)slowly added to 2000 g water, followed by stirring and heating to 80° C.for 1 hour and cooling.

To 88 g of the concentrated PVA solution, 785 g water, 0.88 g SDS, and3.33 g borax is added to form a PVA borax solution.

A monomer emulsion is formed from 7.82 g toluene, 0.040 g2,2′-azobis-(2-methylbutyronitrile) (AMBN), 2.06 g tBDMS HEAM, 0.96 gtBDMS-EBHEAM (95 purity) and 92.9 g PVA borax solution mixed byultraturax for 2 minutes, and further homogenized for 5 minutes.

In a 0.5 L reactor, 1.65 g of a water dispersion of seed particles (seeddiameter 0.319 μm, 8.07 weight % solids) is mixed with 88.34 g of themonomer emulsion. Argon gas (10-20 ml/min) is bubbled through themixture while stirring and heating 1 hour at 30° C. and 2 hours at 40°C. The argon flow is stopped, and heating and stirring continued for 3hours at 80° C.

The reaction mixture is transferred to a 1 liter centrifugation flaskand centrifuged in a Sorvall RC3CPlus centrifuge for 50 minutes at 4500RPM. The creamy flotation product is collected and is centrifuged twicein THF.

To 83.9 g of the THF swollen gel sediment, the same weight of glacialacetic acid and half the weight of water is added. The mixture is shakenat room temperature over night. The gel is worked by removing thesupernatant after centrifugation and adding THF and water in a ratio ofTHF:water 1:1 two times and water once, followed by three times withDMF.

The solids content of the dispersion is determined to be 1.63 g. Thediameter of a water swollen gel can be measured in a microscope withphase contrast equipment and is on average 1.6 μm. The CV is not greaterthan 5.0%.

Example 13

A silyl protected acrylamide monomer,(N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide) (tBDMS-HEAM), ispolymerized withN,N′-(ethane-1,2-diyl)bis(N-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide)(TBDMS EBHEAM) as crosslinker in a dispersed phase formed frompolystyrene particles and is deprotected to form a hydrophilic particle.

A concentrated PVA solution is prepared by adding 80 g polyvinylalcohol(PVA) slowly to 2000 g water while stirring. The mixture is stirred andis heated to 80° C. for 1 hour and cooled.

To 241.8 g of the concentrated PVA solution, 2129.6 g water, 2.32 g SDSand 9.97 g borax are added to form a PVA borax solution.

A monomer emulsion is prepared by mixing 72.68 g toluene, 0.29 g2,2′-azobis-(2-methylbutyronitrile) (AMBN), 14.59 g tBDMS HEAM, 4.46 gTBDMS EBHEAM and 835.8 g PVA borax solution, mixed by ultraturax for 2minutes, and further homogenized for 9 minutes in a high pressureGauline APV-100 homogenizer at 400 Bar.

In a 1 L reactor, 16.86 g of a water dispersion of seed particles (seeddiameter 0.319 μm, 8.07 weight % solids) is mixed with 897 g of themonomer emulsion. The mixture is stirred and heated for 3 hour at 40° C.while bubbling argon through the mixture at 0.05 l/min for the first 2hours and at 0.15 L/min for 1 hour. The argon flow is then stopped andthe emulsion is heated for 3 hours at 80° C.

The reaction mixture is transferred to a 1 liter centrifugation flaskand centrifuged in a Sorvall RC3CPlus centrifuge for 60 minutes at 4700RPM. The creamy flotation product is transferred to a new 500 mL glassflask and resuspended to 370.74 g with water, the pH is adjusted to 3.86with 21.09 g 0.5 M acetic acid. The mixture is stirred at 60° C. for 2hours.

The gel is worked up by adding 9 volumes of THF to the deprotected gel,and centrifuged in a Sorvall RC3CPlus centrifuge for 10 minutes at 3500RPM, followed by two centrifugations with DMF and two centrifugationswith dry DMF, all with addition of 7.5% THF prior to centrifugation. Thesolids content of the gel in DMF is determined to be 6.37 g. The beaddispersion is transferred to water and inspected by microscopy and thebead diameter is 1.7 micron in water.

Example 14

Hydrogel particles formed in accordance with Example 13 are activatedusing methanesulfonyl chloride.

1.51 g of hydroxyl gel from Example 13 dispersed in DMF is divided intotwo centrifugation bottles and washed three times by centrifugation andremoving the supernatant using a solvent mix of 200 ml of anhydrous DMFand 15 g anhydrous THF. After the last centrifugation, the content ofthe two bottles are pooled together and washed once more with anhydrousDMF. The dry content is determined to be 2.32 weight %.

54.74 g of the DMF dispersion containing 2.32 w % hydroxyl gel fromabove is transferred to a three necked round bottom boiling flask withmechanical stirring, before 8.76 g of DMF is added to adjust the drycontent to 2.00%. The round bottom boiling flask is flushed continuouslywith argon. An amount of 0.2077 g of anhydrous pyridine is added,followed by 0.1692 g methanesulfonyl chloride. The flask is stirred atroom temperature over night.

The dispersion is centrifuged with 200 ml anhydrous DMF three times,removing the supernatant after each centrifugation, and is re-suspendedin anhydrous N-methyl-2-pyrrolidone (NMP) to 122.81 g suspension. Thedry content is determined to be 0.91%, giving 1.12 g of activated gelparticles.

Example 15

Approximately 280 million hydrogel particles are prepared as disclosedin Example 11 and subjected to conditions to facilitate duplication ofpolynucleotides present in the disperse phase droplets.

The hydrogel particles are incubated in the presence of a DNA library(L499, approximately 70 million molecules) and Amplitaq Gold® DNAPolymerase (Applied Biosystems) or KOD Hot Start DNA Polymerase (EMDBiosciences). The sample is applied to an Ion OneTouch™ System (LifeTechnologies), templated and enriched essentially according to themanufacturer's instructions. Percentage recovery of hydrogel particlesprior to enrichment is determined using a Guava SYBR Gold Stain system.Enrichment of the hydrogel particles is performed using an Ion OneTouchES™ System (Life Technologies), essentially according to themanufacturer's instructions (Life Technologies). Post-recoverypercentages for hydrogel particles (Table 1) are found to be lower whenusing a KOD polymerase; however, the total post-enrichment recoverypercentages are greater when using a KOD polymerase (27.4%) as comparedto 9.5% and 18.2%, respectively for Amplitaq Gold® DNA polymerase.

TABLE 1 No. of No. of No. of Particles Post-1T Particles Post-ESParticles Recovered Recov- Recovered Recov- Enzyme In (M) Post 1T (M)ery (%) Post-ES (M) ery (%) Amplitaq 280 218 90.0 24.1 9.5 KOD 280 15354.7 42.0 27.4

Example 14

Polymeric particles are prepared in accordance with Example 9,sulfonated to provide 15% mesylation, and treated with acetate andconcentrated ammonia. Following conjugation and PCR, the polymericparticles are utilized in a sequencing test using the Ion Torrent 316chip.

The polymeric particles exhibit a 200q17 value of 1.2M, such as at least1.4 million. Further, the polymeric particles exhibit an aq17 base testof at least 350M, such as at least 400M. In addition, the particlesexhibit a q17 mean of at least of 175, such as at least 180.

Example 15

A silyl protected acrylamide monomer,(N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide) (tBDMS-HEAM), ispolymerized with a tBDMS-EBHEAM crosslinker in a dispersed phase formedfrom polystyrene particles and is deprotected to form a hydrogelparticle.

An emulsion is prepared by first dissolving 1.74 g SDS in 290.00 g waterand then adding 14.50 g acetone and 29.00 g bis(2-ethylhexyl) adipate(DOA). The emulsion is mixed by ultraturax for 2 minutes, and furtherhomogenized for 5.6 minutes in a high pressure Gauline APV-100homogenizer at 400 Bar.

31.14 g of this emulsion is added to 43.89 g of seed particles (seeddiameter 0.140 μm, 4.85 weight % solids) in a flask. The mixture isshaken at 40° C. for 40 h in a shaking bath for activation.

An SDS borax solution is prepared by dissolving 4.54 g SDS and 9.69 gborax to 2369.8 g water.

A monomer emulsion is formed from 125.29 g 2-phenethyl acetate, 0.468 g2,2′-azobis-(2-methylbutyronitrile) (AMBN), 19.51 g tBDMS HEAM, 5.99 gtBDMS-EBHEAM and 816.75 g SDS borax solution mixed by ultraturax for 5minutes, and further homogenized for 9.68 minutes.

In a 1 L reactor, 62.53 g of a water dispersion of activated seedparticles is mixed with 938.1 g of the monomer emulsion. The mixture isstirred and heated at 40° C. for 2 h. The mixture is further stirred andheated at 40° C. for another hour while argon gas (150-200 ml/min) isbubbled through the mixture. The amount of O₂ in the emulsion at thispoint is measure to be 0 ppb. The argon flow is stopped, and heating andstirring continued for 10 hours at 70° C.

The reaction mixture is transferred to four 250 mL centrifugation flasksand centrifuged in a Beckman Coulter Avanti J-20 XP centrifuge for 60minutes at 13000 RPM. The supernatants are discarded and the sedimentsare collected and transferred into a glass flask by adding water.

pH of the aqueous dispersion of gels is adjusted to 3.8 by adding 0.5 Macetic acid solution. The acidified gel dispersion is shaken at 60° C.in a shaking bath for 2 h and cooled.

The gel dispersion is transferred into three 1L flasks, 300 g THF isadded to each, the flasks are shaken at room temperature for 30 min on ashaking table and centrifuged in a Thermo Scientific Thermo ScientificSorvall RC3CPlus centrifuge for 25 minutes at 4500 RPM. The upper phasesof resulting biphasic mixtures are discarded, 50 g THF is added to eachflask, the flasks are shaken at room temperature for 30 min on a shakingtable and centrifuged for 25 minutes at 4500 RPM. Supernatants arediscarded.

Contents of each flask are divided into two 250 mL centrifuge flasks.Approximately 100 g DMF is added on each flask and the flasks are shakenovernight at room temperature on a shaking table. Contents of each flaskare totaled to 200 g by adding 20 g THF and an amount of DMF. The flasksare centrifuged in a Beckman Coulter Avanti J-20 XP centrifuge for 70minutes at 13000 RPM. Supernatants are discarded.

Approximately 100 g DMF is added on each flask and the flasks are shakenfor 40 min at room temperature on a shaking table. Contents of eachflask are totaled to 200 g by adding 20 g THF and an amount of DMF. Theflasks are centrifuged in a Beckman Coulter Avanti J-20 XP centrifugefor 70 minutes at 13000 RPM. Supernatants are discarded and all thesediments are combined into a new flask by using minimal amounts of DMF.

The solids content of the dispersion is determined to be 2.34 g. Thediameter of a water swollen gel is measured in a microscope with phasecontrast equipment and is on average 0.80 μm.

Water swollen gel is further analyzed in a disc centrifuge instrument(CPS Instruments, Inc, model DC20000) using a gradient of 3 and 7 w %sucrose solutions and a rotation speed of 15000 RPM. The diameter ismeasured as 0.4995 μm using a particle density of 1,032 g/ml. CV(number) is measured as 3.6%.

Example 16

A silyl protected acrylamide monomer,(N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide) (tBDMS-HEAM), ispolymerized with a tBDMS-EBHEAM crosslinker in a dispersed phase formedfrom polystyrene particles and is deprotected to form a hydrogelparticle.

An emulsion is prepared by first dissolving 1.98 g SDS in 330.05 g waterand then adding 16.51 g acetone and 33.00 g DOA. The emulsion is mixedby ultraturax for 2 minutes, and further homogenized for 6.4 minutes ina high pressure Gauline APV-100 homogenizer at 400 Bar.

37.71 g of this emulsion is added to 68.57 g of seed particles (seeddiameter 0.081 μm, 4.91 weight % solids) in a flask. The mixture isshaken at 40° C. for 20 h in a shaking bath for activation.

An SDS borax solution is prepared by dissolving 3.77 g SDS and 7.59 gborax to 1975.6 g water.

A monomer emulsion is formed from 33.89 g 2-phenethyl acetate, 0.126 g2,2′-azobis-(2-methylbutyronitrile) (AMBN), 5.26 g tBDMS HEAM, 1.61 gtBDMS-EBHEAM and 223.32 g SDS borax solution mixed by ultraturax for 5minutes, and further homogenized for 2.6 minutes.

In a 250 mL reactor, 20.31 g of a water dispersion of activated seedparticles is mixed with 228.23 g of the monomer emulsion. The mixture isstirred and heated at 40° C. for 2 h. The mixture is further stirred andheated at 40° C. for another hour while argon gas (150-200 ml/min) isbubbled through the mixture. The amount of O₂ in the emulsion at thispoint is measure to be 230 ppb. The argon flow is stopped, and heatingand stirring continued for 10 hours at 70° C.

The reaction mixture is transferred to a 250 mL centrifugation flask andcentrifuged in a Beckman Coulter Avanti J-20 XP centrifuge for 90minutes at 12500 RPM. The supernatant is discarded and the sediment iscollected and transferred into a glass flask by adding water.

pH of the aqueous dispersion of gels is adjusted to 3.85 by adding 0.5 Macetic acid solution. The acidified gel dispersion is shaken at 60° C.in a shaking bath for 2.5 h and cooled.

The gel dispersion is transferred into a 1 L flask, 170.06 g THF isadded, the flask is shaken at room temperature for 10 min on a shakingtable and centrifuged in a Thermo Scientific Thermo Scientific SorvallRC3CPlus centrifuge for 30 minutes at 4500 RPM. The upper phase ofresulting biphasic mixture is discarded. 86.81 g THF is added, the flaskis shaken at room temperature for 15 min on a shaking table andcentrifuged for 30 minutes at 4500 RPM. Supernatant is discarded.

200 g DMF is added on the gel sediment in 1 L flask and this suspensionis divided in two 250 mL centrifuge flasks. The flasks are shaken atroom temperature for 153 min on a shaking table and contents of eachflask are totaled to 200 g by adding 30 g THF and an amount of DMF. Theflasks are centrifuged in a Beckman Coulter Avanti J-20 XP centrifugefor 90 minutes at 14000 RPM. Supernatants are discarded.

100 g DMF is added to each flask and the suspensions are shaken at roomtemperature for 20 min on a shaking table. Contents of each flask aretotaled to 200 g by adding 30 g THF and an amount of DMF and the flasksare centrifuged in a Beckman Coulter Avanti J-20 XP centrifuge for 90minutes at 14000 RPM. Supernatants are discarded and all the sedimentsare combined into a new flask by using minimal amounts of DMF. Thesolids content of the dispersion is determined to be 2.03 g.

Water swollen gel is further analyzed in a disc centrifuge instrument(CPS Instruments, Inc, model DC20000) using a gradient of 3 and 7 w %sucrose solutions and a rotation speed of 20000 RPM. The diameter ismeasured as 0.2885 μm using a particle density of 1.032 g/ml. CV(number) is measured as 5.56%.

Example 17

A protected amino acrylamide monomer(N-tert-butoxycarbonyl-N′-acryloyl-piperazine) and is polymerized withtBDMS-HEAM monomer and tBDMS-EBHEAM crosslinker in a dispersed phaseformed from polystyrene particles and is deprotected to form an aminohydrogel particle.

An emulsion is prepared by first dissolving 1.74 g SDS in 290.00 g waterand then adding 14.50 g acetone and 29.00 g DOA. The emulsion is mixedby ultraturax for 2 minutes, and further homogenized for 5.6 minutes ina high pressure Gauline APV-100 homogenizer at 400 Bar.

41.33 g of this emulsion is added to 62.93 g of seed particles (seeddiameter 0.126 μm, 4.59 weight % solids) in a flask. The mixture isshaken at 40° C. for 40 h in a shaking bath for activation.

An SDS borax solution is prepared by dissolving 4.54 g SDS and 9.69 gborax to 2369.8 g water.

A monomer emulsion is formed from 34.22 g 2-phenethyl acetate, 0.13 gAMBN, 4.98 g tBDMS HEAM, 1.61 g tBDMS-EBHEAM, 0.37 gN-tert-butoxycarbonyl-N′-acryloyl-piperazine and 221.73 g SDS boraxsolution mixed by ultraturax for 5 minutes, and further homogenized for4 minutes.

In a 250 mL reactor, 17.09 g of a water dispersion of activated seedparticles is mixed with 233.1 g of the monomer emulsion. The mixture isstirred and heated at 40° C. for 2 h. The mixture is further stirred andheated at 40° C. for another hour while argon gas (150-200 ml/min) isbubbled through the mixture. The amount of O₂ in the emulsion at thispoint is measure to be 500 ppb. The argon flow is stopped, and heatingand stirring continued for 10 hours at 70° C.

The reaction mixture is transferred to a 250 mL centrifugation flask andcentrifuged in a Beckman Coulter Avanti J-20 XP centrifuge for 60minutes at 12000 RPM. The supernatant is discarded and the sediment iscollected and transferred into a glass flask by adding water.

pH of the aqueous dispersion of gels is adjusted to 3.8 by adding 0.5 Macetic acid solution. The acidified gel dispersion is shaken at 60° C.in a shaking bath for 2.5 h and cooled.

The gel dispersion is transferred into a 1 L flask, 317.24 g THF isadded, the flask is shaken at room temperature for 30 min on a shakingtable and centrifuged in a Thermo Scientific Thermo Scientific SorvallRC3CPlus centrifuge for 30 minutes at 4500 RPM. The upper phase ofresulting biphasic mixture is discarded. 169.63 g THF is added, theflask is shaken at room temperature for 23 min on a shaking table andthe flask is centrifuged for 30 minutes at 4500 RPM. Supernatant isdiscarded.

Approximately 170 g water is added on the gel sediment and the flask isshaken at room temperature overnight on a shaking table. The flask iscentrifuged for 30 minutes at 4500 RPM. Supernatant is discarded.

Approximately 170 g water is added on the gel sediment.

32.2 g of a suspension of the hydrogel (1.55 weight % solids) is dilutedwith 33 g of water. The pH is adjusted to pH 1.0 with 3.2 mL 2M HCl. Thesuspension is transferred to a 250 mL reactor along with 54 g water. Thesuspension is heated at 60 C for 18 hours. The reaction mixture is thentransferred to a 250 mL centrifuge flask in a Beckman Coulter AvantiJ-20 XP.

The gel is worked up by centrifugation after first diluting with waterand titrating the suspension to ca pH 10 with 10 weight % NaOH, giving atotal weight of 175 g. After repeating this, the gel is worked upfurther three times with water. Centrifugation speed is graduallyreduced from 14500 rpm to 6000 rpm for 5 minutes during this process.After discarding the supernatant the gel is then diluted with DMF to 175g, and the obtained suspension is shaken over night. After centrifugingat 6500 rpm for 5 minutes and discarding the supernatant, workup iscontinued with three more corresponding washings with DMF.

The gel is diluted to NMP to 175 g and centrifuged at 7000 rpm for 5minutes. The dry substance is then adjusted to 0.29 weight % solids,yielding 71 g suspension.

Example 18

A protected amino acrylamide monomer(N-fluorenylmethyloxycarbonyl-N′-acryloyl-piperazine) and is polymerizedwith tBDMS-HEAM monomer and tBDMS-EBHEAM crosslinker in a dispersedphase formed from polystyrene particles and is deprotected to form anamino hydrogel particle.

An emulsion is prepared by first dissolving 1.74 g SDS in 290.00 g waterand then adding 14.50 g acetone and 29.00 g DOA. The emulsion is mixedby ultraturax for 2 minutes, and further homogenized for 5.6 minutes ina high pressure Gauline APV-100 homogenizer at 400 Bar.

41.33 g of this emulsion is added to 62.93 g of seed particles (seeddiameter 0.126 μm, 4.59 weight % solids) in a flask. The mixture isshaken at 40° C. for 40 h in a shaking bath for activation.

An SDS borax solution is prepared by dissolving 4.54 g SDS and 9.69 gborax to 2369.8 g water.

A monomer emulsion is formed from 34.22 g 2-phenethyl acetate, 0.13 gAMBN, 4.98 g tBDMS HEAM, 1.61 g tBDMS-EBHEAM, 0.59 gN-fluorenylmethyloxycarbonyl-N′-acryloyl-piperazine and 221.71 g SDSborax solution mixed by ultraturax for 5 minutes, and furtherhomogenized for 4 minutes.

In a 250 mL reactor, 17.09 g of a water dispersion of activated seedparticles is mixed with 233.23 g of the monomer emulsion. The mixture isstirred and heated at 40° C. for 2 h. The mixture is further stirred andheated at 40° C. for another hour while argon gas (150-200 ml/min) isbubbled through the mixture. The amount of O₂ in the emulsion at thispoint is measure to be 62 ppb. The argon flow is stopped, and heatingand stirring continued for 10 hours at 70° C.

The reaction mixture is transferred to a 250 mL centrifugation flask andcentrifuged in a Beckman Coulter Avanti J-20 XP centrifuge for 60minutes at 12000 RPM. The supernatant is discarded and the sediment iscollected and transferred into a glass flask by adding water.

pH of the aqueous dispersion of gels is adjusted to 3.8 by adding 0.5 Macetic acid solution. The acidified gel dispersion is shaken at 60° C.in a shaking bath for 2.5 h and cooled.

The gel dispersion is transferred into a 1 L flask, 287.06 g THF isadded, the flask is shaken at room temperature for 30 min on a shakingtable and centrifuged in a Thermo Scientific Thermo Scientific SorvallRC3CPlus centrifuge for 30 minutes at 4500 RPM. The upper phase ofresulting biphasic mixture is discarded. 111.41 g THF is added, theflask is shaken at room temperature for 23 min on a shaking table andthe flask is centrifuged for 30 minutes at 4500 RPM. Supernatant isdiscarded. DMF is added on the gel and the flask is shaken overnight atroom temperature on a shaking table.

To the suspension of beads in DMF weighing 120 g, 10 mL piperidine isadded and shaken at room temperature on a shaking table for 60 min. 45 gTHF is added and the flask is centrifuged in a Beckman Coulter AvantiJ-20 XP centrifuge for 90 minutes at 14000 RPM. Supernatant isdiscarded.

DMF is added till the suspension weighs 112 g and the gel is shaken on ashaking table for 4 h. 37 g THF is added and the flask is centrifuged ina Beckman Coulter Avanti J-20 XP centrifuge for 60 minutes at 14000 RPM.Supernatant is discarded.

DMF is added till the suspension weighs 130 g and the gel is shaken on ashaking table for 90 min. 40 g THF is added and the flask is centrifugedin a Beckman Coulter Avanti J-20 XP centrifuge for 60 minutes at 14000RPM. Supernatant is discarded.

105 g dry 1-methyl-2-pyrrolidinone (NMP) is added on the batch andshaken for 3 days. The water swollen gel is analyzed in a disccentrifuge instrument (CPS Instruments, Inc, model DC20000) using agradient of 3 and 7 w % sucrose solutions and a rotation speed of 15000RPM. The diameter is measured as 0.3694 μm and CV (number) is measuredas 4.8%.

Example 19

A protected amino acrylamide monomer((9H-fluoren-9-yl)methyl(2-(2-(2-acrylamidoethoxy)ethoxy)ethyl)carbamate)and is polymerized with tBDMS-HEAM monomer and tBDMS-EBHEAM crosslinkerin a dispersed phase formed from polystyrene particles and isdeprotected to form an amino hydrogel particle.

An emulsion is prepared by first dissolving 1.68 g SDS in 280.00 g waterand then adding 14.00 g acetone and 28.00 g DOA. The emulsion is mixedby ultraturax for 2 minutes, and further homogenized for 5.4 minutes ina high pressure Gauline APV-100 homogenizer at 400 Bar.

26.58 g of this emulsion is added to 52.61 g of seed particles (seeddiameter 0.126 μm, 4.59 weight % solids) in a flask. The mixture isshaken at 40° C. for 16 h in a shaking bath for activation.

An SDS borax solution is prepared by dissolving 3.21 g SDS and 8.07 gborax to 1823.6 g water.

A monomer emulsion is formed from 33.68 g 2-phenethyl acetate, 0.13 gAMBN, 4.89 g tBDMS HEAM, 1.61 g tBDMS-EBHEAM, 0.64 g(9H-fluoren-9-yl)methyl(2-(2-(2-acrylamidoethoxy)ethoxy)ethyl)carbamateand 218.95 g SDS borax solution mixed by ultraturax for 5 minutes, andfurther homogenized for 2.6 minutes.

In a 250 mL reactor, 15.53 g of a water dispersion of activated seedparticles is mixed with 234.9 g of the monomer emulsion. The mixture isstirred and heated at 40° C. for 2 h. The mixture is further stirred andheated at 40° C. for another hour while argon gas (150-200 ml/min) isbubbled through the mixture. The amount of O₂ in the emulsion at thispoint is measure to be 75 ppb. The argon flow is stopped, and heatingand stirring continued for 10 hours at 70° C.

The reaction mixture is transferred to a 250 mL centrifugation flask andcentrifuged in a Beckman Coulter Avanti J-20 XP centrifuge for 60minutes at 13000 RPM. The supernatant is discarded and the sediment iscollected and transferred into a glass flask by adding water.

pH of the aqueous dispersion of gels is adjusted to 3.7 by adding 0.5 Macetic acid solution. The acidified gel dispersion is shaken at 60° C.in a shaking bath for 2.5 h and cooled.

The gel dispersion is transferred into a 1 L flask, 180 g THF is addedand centrifuged in a Thermo Scientific Thermo Scientific SorvallRC3CPlus centrifuge for 30 minutes at 4500 RPM. The upper phase ofresulting biphasic mixture is discarded and the mixture is shaken atroom temperature for 15 min on a shaking table. 160 g THF is added andthe flask is centrifuged for 30 minutes at 4500 RPM. Supernatant isdiscarded.

The gel is transferred into a glass flask with DMF. On 50 g of this DMFsuspension of beads, 5 mL piperidine is added and the mixture is shakenat room temperature on a shaking table for 60 min.

50 g of this suspension is transferred to a 250 mL centrifuge flask and23 g THF is added. The flask is centrifuged in a Beckman Coulter AvantiJ-20 XP centrifuge for 10 minutes at 10000 RPM. Supernatant isdiscarded, DMF is added and the mixture is shaken on a shaking table for16 h at room temperature.

Water swollen gel is analyzed in a disc centrifuge instrument (CPSInstruments, Inc, model DC20000) using a gradient of 3 and 7 w % sucrosesolutions and a rotation speed of 15000 RPM. The diameter is measured as0.4517 μm using a particle density of 1.032 g/ml. CV (number) ismeasured as 3.7%.

Example 20

A protected amino acrylamide monomer, N-BocN-acryloyl-4,7,10-trioxatridecane-1,13-diamine, and is polymerized withtBDMS-HEAM monomer and tBDMS-EBHEAM crosslinker in a dispersed phaseformed from polystyrene particles and is deprotected to form an aminohydrogel particle

A PVA solution is prepared by slowly adding 80 g of 87-89% hydrolyzedpolyvinylalcohol (PVA) to 2000 g water, stirring and heating to 80° C.for 1 hour and cooling. An amount of 67.66 g of the PVA solution ismixed with 582.70 g water, 0.62 g SDS and 2.74 g borax.

A monomer emulsion is formed from 26.39 g 2-phenethyl acetate, 0.098 gAMBN, 3.97 g tBDMS HEAM, 1.26 g tBDMS-EBHEAM, 0.21 g N-BocN-acryloyl-4,7,10-trioxatridecane-1,13-diamine and 176.12 g PVA-boraxsolution mixed by ultraturax for few minutes, and further homogenizedfor 2 minutes.

In a 250 mL reactor, 7.15 g of a water dispersion of seed particles(seed diameter 0.319 μm, 8.07 weight % solids) is mixed with 193.02 g ofthe monomer emulsion. The mixture is stirred and heated at 40° C. for 2h. The mixture is further stirred and heated at 40° C. for another hourwhile argon gas (150-200 ml/min) is bubbled through the mixture. Theamount of O₂ in the emulsion at this point is measure to be 40 ppb. Theargon flow is stopped, and heating and stirring continued for 10 hoursat 70° C.

The reaction mixture is transferred to a 250 mL centrifugation flask andcentrifuged in a Beckman Coulter Avanti J-20 XP centrifuge for 40minutes at 12500 RPM. The supernatant is discarded and the sediment iscollected and transferred into a glass flask by adding water.

1 M H₂SO₄ solution is added on the gel suspension in a 9:1 vol ratio toend up in 0.1 M H₂SO₄ concentration. The suspension is shaken at 60° C.for 3 h and cooled down to room temperature.

Concentrated NaOH solution is added dropwise till the pH reaches 12. 500g THF is added to the suspension, divided into two 250 mL bottles andthe mixtures are shaken at room temperature for 60 min on a shakingtable. The flasks are centrifuged in a Thermo Scientific SorvallRC3CPlus centrifuge for 30 minutes at 4500 RPM. The upper phases ofresulting biphasic mixtures are discarded, water is added to each flaskand the flasks are shaken at room temperature overnight on a shakingtable. pH of the mixtures are adjusted to 12.3 by adding concentratedNaOH solution, the flasks are shaken at room temperature for 25 min on ashaking table and the flasks are centrifuged for 30 minutes at 4500 RPMin a Beckman Coulter Avanti J-20 XP centrifuge. Supernatants arediscarded.

Water is added to each flask and pH of the mixtures are adjusted to 12.2by adding concentrated NaOH solution. The flasks are shaken at roomtemperature overnight on a shaking table and the flasks are centrifugedfor 30 minutes at 4000 RPM in a Thermo Scientific Sorvall RC3CPluscentrifuge. Supernatants are discarded.

Water is added to each flask, the flasks are shaken at room temperaturefor 4 h on a shaking table and the flasks are centrifuged for 30 minutesat 6500 RPM in a Beckman Coulter Avanti J-20 XP centrifuge. Supernatantsare discarded

NMP is added to each flask, the flasks are shaken at room temperatureovernight on a shaking table and the flasks are centrifuged for 30minutes at 4500 RPM in a Thermo Scientific Sorvall RC3CPlus centrifuge.Approximately half of supernatants are discarded.

The gel sediments are combined into one bottle, NMP added and the flasksare centrifuged for 60 minutes at 4500 RPM in a Thermo ScientificSorvall RC3CPlus centrifuge. Supernatants are discarded

NMP is added to the flask, the flask is shaken at room temperature for 3h on a shaking table and the flasks are centrifuged for 50 minutes at6500 RPM in a Beckman Coulter Avanti J-20 XP centrifuge. Supernatantsare discarded.

Some more NMP is added and the solids content of the dispersion isdetermined to be 0.108%. The diameter of a water swollen gel is measuredin a microscope with phase contrast equipment and is on average 1.79 μm

Water swollen gel is further analyzed in a disc centrifuge instrument(CPS Instruments, Inc, model DC20000) using a gradient of 3 and 7 w %sucrose solutions and a rotation speed of 10050 RPM. The diameter ismeasured as 0.934 μm using a particle density of 1.032 g/ml.

Example 21

A protected amino acrylamide monomer, N-BocN-acryloyl-4,7,10-trioxatridecane-1,13-diamine, and is polymerized withtBDMS-HEAM monomer and tBDMS-EBHEAM crosslinker in a dispersed phaseformed from polystyrene particles and is deprotected to form an aminohydrogel particle

An emulsion is prepared by first dissolving 1.74 g SDS in 290.00 g waterand then adding 14.50 g acetone and 29.00 g bis(2-ethylhexyl) adipate(DOA). The emulsion is mixed by ultraturax for 2 minutes, and furtherhomogenized for 5.6 minutes in a high pressure Gauline APV-100homogenizer at 400 Bar.

26.58 g of this emulsion is added to 52.27 g of seed particles (seeddiameter 0.126 μm, 4.59 weight % solids) in a flask. The mixture isshaken at 40° C. for 40 h in a shaking bath for activation.

An SDS borax solution is prepared by dissolving 1.87 g SDS and 4.04 gborax to 987.5 g water.

A monomer emulsion is formed from 62.81 g 2-phenethyl acetate, 0.238 gAMBN, 9.13 g tBDMS HEAM, 3.00 g tBDMS-EBHEAM, 1.05 g N-BocN-acryloyl-4,7,10-trioxatridecane-1,13-diamine and 408.39 g SDS-boraxsolution mixed by ultraturax for few minutes, and further homogenizedfor 4.8 minutes.

In a 500 mL reactor, 31.06 g of a water dispersion of activated seedparticles is mixed with 469.6 g of the monomer emulsion. The mixture isstirred and heated at 40° C. for 2 h. The mixture is further stirred andheated at 40° C. for another hour while argon gas (200 ml/min) isbubbled through the mixture. The argon flow is stopped, and heating andstirring continued for 10 hours at 70° C.

The reaction mixture is transferred to four 250 mL centrifugation flasksand centrifuged in a Beckman Coulter Avanti J-20 XP centrifuge for 60minutes at 13000 RPM. The supernatants are discarded and the sedimentsare collected and transferred into a glass flask by adding water.

pH of the aqueous dispersion of gels is adjusted to 3.8 by adding 0.5 Macetic acid solution. The acidified gel dispersion is shaken at 60° C.in a shaking bath for 2 h and cooled.

1 M H₂SO₄ solution is added on the gel suspension in a 9:1 vol ratio toend up in 0.1 M H₂SO₄ concentration. The suspension is shaken at 60° C.for 3 h and cooled down to room temperature.

Concentrated NaOH solution is added dropwise till the pH reaches 12. Thesuspension is divided into two 1 L centrifuge flasks. 380 g THF is addedto each flask, and the mixtures are shaken at room temperature for 145min on a shaking table. The flasks are centrifuged in a ThermoScientific Sorvall RC3CPlus centrifuge for 30 minutes at 4500 RPM.Supernatants are discarded, water is added to each flask and the flasksare shaken at room temperature for 2 h on a shaking table. More water isadded and the flasks are centrifuged in a Thermo Scientific SorvallRC3CPlus centrifuge for 60 minutes at 4500 RPM. Supernatants arediscarded.

The gel is transferred into four 250 mL centrifuge flasks and the flasksare centrifuged for 45 minutes at 14500 RPM in a Beckman Coulter AvantiJ-20 XP centrifuge. Supernatants are discarded.

Water is added to each flask, the flasks are shaken at room temperaturefor 60 min on a shaking table and the flasks are centrifuged for 45minutes at 14500 RPM in a Beckman Coulter Avanti J-20 XP centrifuge.Supernatants are discarded.

Water is added to each flask and pH of the mixtures are adjusted to 12by adding concentrated NaOH solution. The flasks are shaken at roomtemperature for 25 min on a shaking table and the flasks are centrifugedfor 30 minutes at 6500 RPM in a Beckman Coulter Avanti J-20 XPcentrifuge. Supernatants are discarded.

Water is added to each flask and pH of the mixtures are adjusted to 12.3by adding concentrated NaOH solution. The flasks are shaken at roomtemperature overnight on a shaking table and the flasks are centrifugedfor 30 minutes at 4000 RPM in a Thermo Scientific Sorvall RC3CPluscentrifuge. Supernatants are discarded.

Water is added to each flask, the flasks are shaken at room temperaturefor 4 h on a shaking table and the flasks are centrifuged for 30 minutesat 6500 RPM in a Beckman Coulter Avanti J-20 XP centrifuge. Supernatantsare discarded

NMP is added to each flask, the flasks are shaken at room temperatureovernight on a shaking table and the flasks are centrifuged for 30minutes at 4500 RPM in a Thermo Scientific Sorvall RC3CPlus centrifuge.Supernatants are discarded.

NMP is added to the flasks, the flasks are shaken at room temperaturefor 5 h on a shaking table and the flasks are centrifuged for 50 minutesat 6500 RPM in a Beckman Coulter Avanti J-20 XP centrifuge. Supernatantsare discarded.

Some more NMP is added and the solids content of the dispersion isdetermined to be 0,408%. The diameter of a water swollen gel is measuredin a microscope with phase contrast equipment and is on average 0.86 μm.The diameter of a water swollen gel is also measured in disc centrifugeinstrument (CPS Instruments, Inc, model DC20000) using a gradient of 3and 7 w % sucrose solutions and a rotation speed of 15000 RPM. Thediameter is measured as 0.511 μm using a particle density of 1.032 g/ml.CV (number) is measured as 4.82%.

Example 22

tBDMS-HEAM is polymerized with tBDMS-EBHEAM crosslinker in a dispersedphase formed from polystyrene particles and is deprotected to form ahydrogel particle.

An emulsion is prepared by first dissolving 1.14 g SDS in 190.00 g waterand then adding 28.50 g acetone and 19.00 g DOA. The emulsion is mixedby ultraturax for 5 minutes, and further homogenized for 4 minutes in ahigh pressure Gauline APV-100 homogenizer at 400 Bar.

30.55 g of this emulsion is added to 7.39 g of seed particles (seeddiameter 4.96 μm, 9.54 weight % solids) in a flask. The mixture isshaken at 40° C. for 22 h in a shaking bath for activation.

300 g H₂O is heated up to 80° C. and 4.2 g Methocel K-100 is dissolvedin. 332 g H₂O is added to obtain the Methocel K-100 solution.

2.41 g borax is added onto 94.8 g Methocel solution. The weight of thissolution is totaled up to 420.2 g by adding water to obtain the boraxsolution is prepared by mixing

A monomer emulsion is formed from 35.72 g 2-phenethyl acetate, 0.138 gAMBN, 5.56 g tBDMS HEAM, 1.71 g tBDMS-EBHEAM and 165.89 g borax solutionmixed by ultraturax for few minutes, and further homogenized for 2.1minutes.

In a 250 mL reactor, 9.25 g of a water dispersion of activated seedparticles is mixed with 177.0 g of the monomer emulsion and 63.26 gMethocel K-100 solution. The mixture is stirred and heated at 40° C. for2.5 h. The mixture is further stirred and heated at 40° C. for another30 min while argon gas (200 ml/min) is bubbled through the mixture. Theargon flow is stopped, and heating and stirring continued for 10 hoursat 70° C.

Part of the reaction mixture is transferred to a 250 mL centrifugationflask and centrifuged in a Beckman Coulter Avanti J-20 XP centrifuge for60 minutes at 13000 RPM. The supernatant is discarded and the sedimentis collected and transferred into a glass flask by adding water.

pH of the aqueous dispersion of gels is adjusted to 3.8 by adding 0.5 Macetic acid solution. The acidified gel dispersion is shaken at 60° C.in a shaking bath for 150 min and cooled.

The gel dispersion is transferred into a 1 L flask, 300 g THF is added,and the flask is shaken at room temperature for 30 min on a shakingtable and centrifuged in a Thermo Scientific Sorvall RC3CPlus centrifugefor 25 minutes at 4500 RPM. The upper phase of resulting biphasicmixture is discarded, 50 g THF is added to the flask. The flask isshaken at room temperature for 30 min on a shaking table and centrifugedfor 25 minutes at 4500 RPM. Supernatant is discarded.

Contents of the flask are divided into two 250 mL centrifuge flasks.Approximately 100 g DMF is added on each flask and the flasks are shakenovernight at room temperature on a shaking table. Contents of each flaskare totaled to 200 g by adding 20 g THF and an amount of DMF. The flasksare centrifuged in a Beckman Coulter Avanti J-20 XP centrifuge for 70minutes at 13000 RPM. Supernatants are discarded.

Approximately 100 g DMF is added on each flask and the flasks are shakenfor 40 min at room temperature on a shaking table. Contents of eachflask are totaled to 200 g by adding 20 g THF and an amount of DMF. Theflasks are centrifuged in a Beckman Coulter Avanti J-20 XP centrifugefor 70 minutes at 13000 RPM. Supernatants are discarded and all thesediments are combined into a new flask by using minimal amounts of DMF.

The solids content of the dispersion is determined to be 2.34 g. Thediameter of water swollen gels is measured in a microscope with phasecontrast equipment and is on average 47.5 μm.

Example 23

tBDMS-HEAM monomer is polymerized in an aqueous emulsion to formmonosized seed particles.

250 mL of water is boiled in a 250 mL conical flask for 10 minutes andcooled down with ice-water bath while purging Ar gas into.

In a 50 mL conical flask, 0.04 g SDS is dissolved in 10.1 g of thisboiled water by using a magnetic stirbar.

In another 50 mL conical flask, 0.059 g potassium persulfate isdissolved in 10 g of the boiled water by using a magnetic stirbar.

Into a 100 mL jacketed two-piece reactor equipped with a mechanicalstirrer, a temperature probe, a water running condenser and an Arsource, 0.20 g borax and 80 g of boiling water, boiled previously for atleast 30 min, are added and the reactor is heated to 80° C. by using aheating bath while the overhead stirrer equipped with apoly(tetrafluoroethylene) blade is set to stir at 150±5 RPM. Thesolution is purged with Ar for 20 min.

When the temperature of the solution reaches 80° C., the Ar tubing islifted out of the solution with Ar pressure is still on and the SDSsolution, ultrasonicated for 5 min right before, is added into thereactor.

8 min after the addition of SDS solution, the mechanical stirrer is setto stir at 250±5 RPM.

9.22 g tBDMS-HEAM is purged with Ar for 5 min and then added into thereactor. 1 min after the addition of tBDMS-HEAM, the mechanical stirreris set to stir at 350±5 RPM.

5 min after the addition of tBDMS-HEAM, potassium persulfate solution,purged with Ar for 5 min right before, is added quickly into the reactorand the reactor is sealed while Ar flow is still running above theemulsion.

270 min after the addition of potassium persulfate, mechanical stirreris set to stir at 250±5 RPM and the Ar flow is stopped. The emulsion isfurther polymerized for 18 h.

The batch is cooled down to room temperature and the whole batch istransferred into a plastic bottle. The diameter is measured by dynamiclight scattering (Malvern, Nano ZS) and found to be 0.398 m. PDI ismeasured to be 0.015. Weight average molecular weight and number averagemolecular weights are measured to be 707 kDa and 102 kDa, respectively,by using a gel permeation chromatography instrument (Waters 717plusequipped with a Waters 2414 refractive index detector and PolymerLaboratories 5 μm Mixed-C 300 mm×7.5 mm columns).

The CV of the particle is measured in disc centrifuge instrument (CPSInstruments, Inc, model DC20000) using a gradient of 3 and 7 w % sucrosesolutions and a rotation speed of 20 000 RPM and found to be 2.0%.

Example 24

Activation of an amino-hydrogel and conjugation of the hydrogel withamine terminal DNA probe.

To a solution of 100 billion of amino-hydrogel (diameter=0.55 microns,23 million amines/micron3) in anhydrous, amine-free N-methylpyrrolidone(NMP) (600 μL), solid bis-succinimidyl suberate (22.1 mg, 60 μmole) isadded followed by tributylamine (14 μL, 60 μmole). After stirring at 60C for 1 h, the hydrogels are isolated by centrifugation (30 min at 21300rcf). The hydrogel pellet is diluted with amine-free anhydrous NMP (1ml) and is isolated by centrifugation; this washing process is repeated2 times, and the final pellet is re-suspended in NMP (600 μL). Thishydrogel suspension is treated with acetic anhydride (30 μL, 317 μmole)and tributylamine (30 μL, 126 μmole), and stirred at room temperaturefor 2 h. The resulting hydrogel is isolated by centrifugation (30 min at21300 rcf) and the pellet is diluted with amine-free anhydrous NMP (1ml) and is isolated by centrifugation; this washing process is repeated2 times, and the final activated, capped pellet is diluted with 1 □μmoleof a 3 molar NMP solution of tetrabutylammonium5′-amino-oligonucleotide, tributylamine (1 μmole), and amine-free NMP toa final volume of 600 After stirring at 70° C. for 16 h, the DNAconjugated hydrogel is isolated by centrifugation (30 min at 21300 rcf).The pellet is washed with NMP (1 ml), followed by Deionized water wash(1 ml) using centrifugation to isolate the pellets. The final hydrogelpellet is diluted with 1×TE buffer (1.6 ml) and stirred at 80 C for 1 h.The hydrogels are isolated by centrifugation (30 min at 21300 rcf) andwashed twice with DI water (1 ml) (using centrifugation for pelletisolation). To the final pellet is added 30% aqueous ammonia; after 15minutes at room temperature, the hydrogel is isolated centrifugation (20min at 21300 rcf) and is washed 3× with DI water (1 mL) usingcentrifugation for isolation. The final pellet is re-dispersed in thebuffer desired for performing target amplification.

Example 25

An oligonucleotide is directly conjugated to a mesyl activated particle.Mesyl chloride activated microgels are prepared via seeded emulsionpolymerization. Thus formed particles are washed inN-methyl-2-pyrrolidone (NMP) in preparation for conjugation with ionexchanged single stranded DNA.

The sodium salt of 5′-NH2-C6-30-mer oligonucleotide was dissolved in 0.1M tetrabutylammonium acetate, and injected onto a reverse phase HPLCcolumn. Elution was performed with 0.1 M tetrabutylammonium acetatemobile phase. The fraction containing nucleic acid was collected,lyophilized to a dry powder, and re-suspended in dryN-methyl-2-pyrrolidone (NMP).

Five million particles (5.0×10⁹) are dispersed in 350 uL of anhydrousNMP and vortex mixed to disperse. 124 uL of Bu4NAc-DNA (5′-NH2-C6-30-meroligonucleotide) in NMP (4.10 mM) is directly added to the particlemixture. 19.5 uL of tetraethylammonium borate (26.14 mM) is then addedto the reaction mixture for a final volume of ˜500 uL.

The reaction mixture is quickly vortex mixed and is gently mixed at 70°C. for 16 hours. The mixture is centrifuged, the supernatant isdecanted, and the particles are re-suspended in 1 mL of NMP. Aftervortex mixing, the re-suspended microgel particles are pelleted with twocycles of precipitation/dispersion in NMP. After the second NMP wash,the pellets are brought up in 1 mL of 2×SSPE/0.1% sodium dodecyl sulfate(SDS), mixed and centrifuged to pellets. Finally, the particles arebrought up in 1 mL of 1×PBS/0.1% Triton X-100, mixed and centrifuged toa firm pellet, repeating this process three times. After the finalcycle, the conjugated microgels are re-suspended in 500 uL 1×PBS/0.1%Triton X-100.

In a first aspect, a method of forming a particle includes, in adisperse phase within an aqueous suspension, polymerizing a plurality ofmer units of a hydrophilic monomer having a hydrophobic protectiongroup, thereby forming a polymeric particle including a plurality of thehydrophobic protection groups, and converting the polymeric particle toa hydrogel particle.

In an example of the first aspect, converting the polymeric particleincludes removing at least a portion of the plurality of the hydrophobicprotection groups from the polymeric particle. For example, removing atleast a portion of the plurality of the hydrophobic protection groupsincludes acid cleaving at least a portion of the plurality of thehydrophobic protection groups from the polymeric particle.

In another example of the first aspect and the above examples,converting the polymeric particle includes removing substantially all ofthe plurality of hydrophobic protection groups from the polymericparticle.

In a further example of the first aspect and the above examples, themethod further includes promoting a seed particle in the aqueoussuspension to form the dispersed phase. In an example, the mass ratio ofprotected monomer:seed particles is in a range of 150:1 to 1:1, such asa range of 50:1 to 1:1. In another example, the seed particle includes aseed polymer. The method can further include extracting the seed polymerafter converting the polymeric particle. In an example, the seed polymercan be hydrophobic. In another example, the seed polymer includes astyrenic polymer, an acrylic polymer, another vinyl polymer, or acombination thereof. In a particular example of the above examples, theseed particle has an initial particle size of not greater than 0.6micrometers, such as not greater than 0.45 micrometers, not greater than0.35 micrometers, or not greater than 0.15 micrometers. In anotherexample of the above examples, promoting the seed particle includesmixing a solvent and a promoting agent with the seed particle. Forexample, the promoting agent is hydrophobic. In another example, thepromoting agent includes dioctanoyl peroxide.

In an additional example of the first aspect and the above examples, thehydrophilic monomer includes an acrylamide.

In another example of the first aspect and the above examples, thehydrophobic protection group includes a hydroxyl protection group.

In a further example of the first aspect or the above examples, thehydrophobic protection group includes an organometallic moiety. Forexample, the organometallic moiety forms a silyl ether functional group.In an example, the silyl ether function group is derived fromtert-butyldimethylsilane ether, trimethylsilyl ether, triethylsilylether, diphenyl methyl silyl ether, or a combination thereof.

In an example of the first aspect and the above examples, polymerizingthe plurality of mer units further includes mixing a crosslinker withthe hydrophilic monomer having a hydrophobic protection group. Forexample, mixing the crosslinker can include mixing the crosslinker at amass ratio of hydrophilic monomer:crosslinker in a range of 15:1 to 1:2.The range can be 10:1 to 1:1. In another example, the crosslinker is alow water solubility crosslinker. In an example of the above examples,the crosslinker is a divinyl crosslinker. For example, the divinylcrosslinker includes a diacrylamide. In a particular example of theabove examples, the diacrylamide includesN,N′-(ethane-1,2-diyl)bis(N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide,N,N′-(2-hydroxypropane-1,3-diyl)diacrylamide, a protected derivativethereof, or a combination thereof. The diacrylamide can, for example,includeN,N′-(ethane-1,2-diyl)bis(N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide,N,N′-(N-(2-((tert-butyldimethylsilyl)oxy)propane-1,3-diyl)diacrylamide,N,N′-(ethane-1,2-diyl)bis(N-(2-((triethylsilyl)oxy)ethyl)acrylamide,N,N′-(N-(2-((triethylsilyl)oxy)propane-1,3-diyl)diacrylamide,silyl-protected N-[2-(acryloylamino)-1,2-dihydroxyethyl]acrylamide suchas N,N′(2,3-bis((triethylsilyl)oxy)butane-1,4-diyl)diacrylamide, or acombination thereof. In another example of the above examples, thedivinyl crosslinker includes ethyleneglycoldimethacrylate,divinylbenzene, hexamethylene bisacrylamide, trimethylolpropanetrimethacrylate, or a combination thereof.

In another example of the first aspect and the above examples,polymerizing the plurality of mer units includes mixing a porogen withthe hydrophilic monomer having a hydrophobic protection group. Forexample, the porogen can be an aromatic porogen. In an example, thearomatic porogen includes toluene.

In an additional example of the first aspect and the above examples, themethod further includes activating the hydrogel particle. In an example,converting includes providing one or more hydroxyl groups on thehydrogel particle and wherein activating includes converting at leastone of the one or more hydroxyl groups to a sulfonate ester group. Forexample, converting can include providing one or more hydroxyl groups onthe hydrogel particle and wherein activating includes replacing at leastone of the one or more hydroxyl groups with an azide functional moiety.In another example, the method further includes binding anoligonucleotide to the activated hydrogel polymer. For example, bindingincludes nucleophilic substitution and the oligonucleotide is anucleophile-terminated oligonucleotide. A nucleophile of thenucleophile-terminated oligonucleotide can be an amine group. Anucleophile of the nucleophile-terminated oligonucleotide can be a thiolgroup. In an example of the above example, the method further includeshybridizing a polynucleotide to the oligonucleotide. For example, themethod can further include amplifying the polynucleotide into aplurality of polynucleotides and attaching at least a portion of theplurality of polynucleotides to the hydrogel particle, therebygenerating a hydrogel particle including a plurality of attachedpolynucleotides. Alternatively, the method can further includeamplifying the polynucleotide into a plurality of complementarypolynucleotides by extending the oligonucleotide, thereby generating ahydrogel particle including a plurality of attached polynucleotides.

In another example of the first aspect and the above examples, thehydrogel particle is one of a plurality of similarly formed hydrogelparticles having an average particle size of not greater than 2micrometer. For example, the average particle size can be not greaterthan 1 micrometer, such as not greater than 0.8 micrometers, or notgreater than 0.5 micrometers.

In a further example of the first aspect and the above examples, thehydrogel particle is one of a plurality of similarly formed hydrogelparticles that are substantially uniform in size.

In an additional example of the first aspect or the above examples, thehydrogel particle is one of a plurality of similarly formed hydrogelparticles having a coefficient of variance of not greater than 5.0%. Forexample, the coefficient of variance of not greater than 3.5%.

In a second aspect, a method of forming a particle includes, in adisperse phase within an aqueous suspension, polymerizing a plurality ofmer units of an acrylamide monomer having a hydrophobic protectiongroup, thereby forming a polymeric particle including a plurality of thehydrophobic protection groups, and converting the polymeric particle toa hydrophilic particle.

In an example of the second aspect, converting the polymeric particleincludes removing at least a portion of the plurality of the hydrophobicprotection groups from the polymeric particle. For example, removing atleast a portion of the plurality of the hydrophobic protection groupsincludes acid cleaving at least a portion of the plurality of thehydrophobic protection groups from the polymeric particle.

In another example of the second aspect or the above examples,converting the polymeric particle includes removing substantially all ofthe plurality of hydrophobic protection groups from the polymericparticle.

In an additional example of the second aspect or the above examples, themethod further includes promoting a seed particle in the aqueoussuspension to form the dispersed phase. For example, the mass ratio ofprotected monomer:seed particles is in a range of 50:1 to 1:1. Inanother example, the seed particle includes a seed polymer. In anexample of the above examples, the method further includes extractingthe seed polymer after converting the polymeric particle. The seedpolymer can be hydrophobic. In another example, the seed polymerincludes a styrenic polymer, an acrylic polymer, another vinyl polymer,or a combination thereof. In an example of the above examples, the seedparticle has an initial particle size of not greater than 0.6micrometers. In an additional example of the above examples, promotingthe seed particle includes mixing a solvent and a promoting agent withthe seed particle. For example, the promoting agent can be hydrophobic.

In another example of the second aspect or the above examples, thehydrophobic protection group includes a hydroxyl protection group. In anadditional example of the second aspect and the above examples, thehydrophobic protection group includes an organometallic moiety. Forexample, the organometallic moiety can form a silyl ether functionalgroup. In an example, the silyl ether function group can be derived fromtert-butyldimethylsilane ether, trimethylsilyl ether, triethylsilylether, diphenyl methyl silyl ether, or a combination thereof.

In a further example of the second aspect and the above examples,polymerizing the plurality of mer units further includes mixing acrosslinker with the acrylamide monomer having a hydrophobic protectiongroup. Mixing the crosslinker can include mixing the crosslinker at amass ratio of hydrophilic monomer:crosslinker in a range of 15:1 to 1:2.In an example, the crosslinker is a low water solubility crosslinker. Inanother example, the crosslinker is a divinyl crosslinker.

In an additional example of the second aspect and the above examples,polymerizing the plurality of mer units includes mixing a porogen withthe acrylamide monomer having a hydrophobic protection group. Forexample, the porogen can include an aromatic porogen.

In another example of the second aspect and the above examples, themethod further includes activating the hydrophilic particle. In anexample, converting includes providing one or more hydroxyl groups onthe hydrogel particle and wherein activating includes converting atleast one of the one or more hydroxyl groups to a sulfonate ester group.In another example, converting includes providing one or more hydroxylgroups on the hydrogel particle and wherein activating includesreplacing at least one of the one or more hydroxyl groups with an azidefunctional moiety. In an additional example, the method further includesbinding an oligonucleotide to the activated hydrogel polymer. Bindingcan include nucleophilic substitution and the oligonucleotide is anucleophile-terminated oligonucleotide. A nucleophile of thenucleophile-terminated oligonucleotide can be an amine group. In anotherexample, a nucleophile of the nucleophile-terminated oligonucleotide caninclude a thiol group. In an additional example, the method furtherincludes hybridizing a polynucleotide to the oligonucleotide. In anotherexample, the method further includes amplifying the polynucleotide intoa plurality of polynucleotides and attaching at least a portion of theplurality of polynucleotides to the hydrophilic particle, therebygenerating a hydrophilic particle including a plurality of attachedpolynucleotides. Alternatively, the method can further includeamplifying the polynucleotide into a plurality of complementarypolynucleotides by extending the oligonucleotide, thereby generating ahydrogel particle including a plurality of attached polynucleotides.

In a further example of the second aspect and the above examples, thehydrophilic particle is one of a plurality of similarly formedhydrophilic particles having an average particle size of not greaterthan 2 micrometer.

In an additional example of the second aspect and the above examples,the hydrophilic particle is one of a plurality of similarly formedhydrophilic particles that are substantially uniform in size.

In another example of the second aspect and the above examples, thehydrophilic particle is one of a plurality of similarly formedhydrophilic particles having a coefficient of variance of not greaterthan 5.0%.

In a third aspect, a method of forming a particle includes, in adisperse phase within an aqueous suspension, polymerizing a plurality ofmer units of an radically polymerizable monomer with a diacrylamidecrosslinker having a hydrophobic protection group, thereby forming apolymeric particle including a plurality of the hydrophobic protectiongroups. The method further includes removing at least a portion ofplurality of the hydrophobic protection groups.

In an example of the third aspect, the diacrylamide includesN,N′-(ethane-1,2-diyl)bis(N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide,N,N′-(2-hydroxypropane-1,3-diyl)diacrylamide, a protected derivativethereof, or a combination thereof. The diacrylamide can, for example,includeN,N′-(ethane-1,2-diyl)bis(N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide,N,N′-(N-(2-((tert-butyldimethylsilyl)oxy)propane-1,3-diyl)diacrylamide,N,N′-(ethane-1,2-diyl)bis(N-(2-((triethylsilyl)oxy)ethyl)acrylamide,N,N′-(N-(2-((triethylsilyl)oxy)propane-1,3-diyl)diacrylamide,silyl-protected N-[2-(acryloylamino)-1,2-dihydroxyethyl]acrylamide suchas N,N′(2,3-bis((triethylsilyl)oxy)butane-1,4-diyl)diacrylamide, or acombination thereof.

In another example of the third aspect or the above examples,polymerizing includes mixing the diacrylamide crosslinker at a massratio of radically polymerizable:crosslinker in a range of 15:1 to 1:2.

In an additional example of the third aspect or the above examples,removing at least a portion of the plurality of the hydrophobicprotection groups includes acid cleaving at least a portion of theplurality of the hydrophobic protection groups from the polymericparticle.

In a further example of the third aspect or the above examples, theradically polymerizable monomer is a vinyl-based monomer. In an example,the vinyl-based monomer includes an acrylate, an acrylamide, a vinylalcohol, a vinyl acetate, acrylamido-methyl-propanesulfonic acid, or acombination thereof. For example, the vinyl-based monomer is anacrylamide.

In another example of the third aspect or the above examples, the methodfurther includes promoting a seed particle in the aqueous suspension toform the dispersed phase. For example, the mass ratio of protectedmonomer:seed particles is in a range of 50:1 to 1:1. In another example,the seed particle includes a seed polymer. In a further example, themethod further includes extracting the seed polymer after converting thepolymeric particle. In an example, the seed polymer is hydrophobic. Inanother example, the seed polymer includes a styrenic polymer, anacrylic polymer, another vinyl polymer, or a combination thereof. In anadditional example, the seed particle has an initial particle size ofnot greater than 0.6 micrometers. In another example, promoting the seedparticle includes mixing a solvent and a promoting agent with the seedparticle.

In a further example of the third aspect or the above examples, thehydrophobic protection group includes a hydroxyl protection group.

In an additional example of the third aspect or the above examples, thehydrophobic protection group includes an organometallic moiety. In anexample, the organometallic moiety forms a silyl ether functional group.In another example, the silyl ether function group is derived fromtert-butyldimethylsilane ether, trimethylsilyl ether, triethylsilylether, diphenyl methyl silyl ether, or a combination thereof.

In another example of the third aspect or the above examples,polymerizing the plurality of mer units includes mixing a porogen withthe radically polymerizable monomer and the diacrylamide crosslinker.For example, the porogen can be an aromatic porogen.

In a further example of the third aspect or the above examples, themethod further includes activating the hydrophilic particle. Forexample, the method further includes binding an oligonucleotide to theactivated hydrogel polymer. In another example, binding includesnucleophilic substitution and the oligonucleotide is anucleophile-terminated oligonucleotide. For example, a nucleophile ofthe nucleophile-terminated oligonucleotide is an amine group. In afurther example, a nucleophile of the nucleophile-terminatedoligonucleotide is a thiol group. In another example, the method furtherincludes hybridizing a polynucleotide to the oligonucleotide. In anadditional example, the method further includes amplifying thepolynucleotide into a plurality of polynucleotides and attaching atleast a portion of the plurality of polynucleotides to the hydrophilicparticle, thereby generating a hydrophilic particle including aplurality of attached polynucleotides. Alternatively, the method canfurther include amplifying the polynucleotide into a plurality ofcomplementary polynucleotides by extending the oligonucleotide, therebygenerating a hydrogel particle including a plurality of attachedpolynucleotides.

In an additional example of the third aspect or the above examples, thehydrophilic particle is one of a plurality of similarly formedhydrophilic particles having an average particle size of not greaterthan 2 micrometer.

In another example of the third aspect or the above examples, thehydrophilic particle is one of a plurality of similarly formedhydrophilic particles having a coefficient of variance of not greaterthan 5.0%.

In a fourth aspect, a method of forming a particle includes polymerizinga plurality of mer units of a hydrophilic monomer having a hydrophobicprotection group, thereby forming a polymeric particle including aplurality of the hydrophobic protection groups; removing at least aportion of plurality of the hydrophobic protection groups from thepolymeric particle to form a hydrophilic particle; and binding anoligonucleotide to the hydrophilic particle.

In an example of the fourth aspect or the above examples, removing atleast a portion of the plurality of the hydrophobic protection groupsincludes acid cleaving at least a portion of the plurality of thehydrophobic protection groups from the polymeric particle.

In another example of the fourth aspect or the above examples, thehydrophilic monomer includes an acrylamide.

In an additional example of the fourth aspect or the above examples, thehydrophobic protection group includes a hydroxyl protection group.

In a further example of the fourth aspect or the above examples, thehydrophobic protection group includes an organometallic moiety. Forexample, the organometallic moiety can form a silyl ether functionalgroup.

In an example of the fourth aspect or the above examples, polymerizingthe plurality of mer units further includes mixing a crosslinker withthe hydrophilic monomer having a hydrophobic protection group. Forexample, the crosslinker can be a divinyl crosslinker. In anotherexample, the divinyl crosslinker includes a diacrylamide.

In another example of the fourth aspect or the above examples, themethod further includes activating the hydrogel particle prior tobinding the oligonucleotide. For example, removing can includesproviding one or more hydroxyl groups on the hydrophilic particle andwherein activating includes converting at least one of the one or morehydroxyl groups to a sulfonate ester group. In another example, removingincludes providing one or more hydroxyl groups on the hydrophilicparticle and wherein activating includes replacing at least one of theone or more hydroxyl groups with an azide functional moiety. In anadditional example, binding includes binding the oligonucleotide to theactivated hydrogel polymer. In an additional example, binding includesnucleophilic substitution and the oligonucleotide is anucleophile-terminated oligonucleotide. For example, a nucleophile ofthe nucleophile-terminated oligonucleotide can be an amine group. Inanother example, a nucleophile of the nucleophile-terminatedoligonucleotide can include a thiol group.

In a further example of the fourth aspect or the above examples, themethod further includes hybridizing a polynucleotide to theoligonucleotide. For example, the method further includes amplifying thepolynucleotide into a plurality of polynucleotides and attaching atleast a portion of the plurality of polynucleotides to the hydrogelparticle, thereby generating a hydrogel particle including a pluralityof attached polynucleotides. Alternatively, the method can furtherinclude amplifying the polynucleotide into a plurality of complementarypolynucleotides by extending the oligonucleotide, thereby generating ahydrogel particle including a plurality of attached polynucleotides.

In a fifth aspect, a plurality of particles includes at least 100,000particles. At least one particle of the plurality of particles includesa hydrogel. The plurality of particles have an average particle size ofnot greater than 100 micrometers and a coefficient of variance of notgreater than 5%. For example, the coefficient of variance is not greaterthan 4.5%, such as not greater than 4.0%, not greater than 3.5%, or notgreater than 3.0%.

In an example of the fifth aspect or the above examples, the averagesize is not greater than 30 micrometers, such as not greater than 1.5micrometers, not greater than 1.1 micrometers, not greater than 0.6micrometers, or not greater than 0.5 micrometers.

In another example of the fifth aspect or the above examples, thehydrogel includes an acrylamide polymer.

In a further example of the fifth aspect or the above examples, theparticles of the plurality of particles have an average porosity of atleast 60%.

In a sixth aspect, a system includes an array of wells. At least onewell of the array of wells is operatively connected with an ISFETsensor. The system further includes a plurality of hydrogel particleshaving a coefficient of variance of not greater than 5%. At least one ofthe hydrogel particles of the plurality of hydrogel particles isdisposed in a well of the array of wells.

In a seventh aspect, a plurality of particles is formed by the methodincluding, in a disperse phase within an aqueous suspension,polymerizing a plurality of mer units of a hydrophilic monomer having ahydrophobic protection group, thereby forming a polymeric particleincluding a plurality of the hydrophobic protection groups, andincluding converting the polymeric particle to a hydrogel particle.

In an example of the seventh aspect, the plurality of particles have acoefficient of variance of not greater than 5.0%, such as not greaterthan 4.0%, not greater than 3.5%, or not greater than 3.0%.

In another example of the seventh aspect or the above examples, theplurality of particles having an average size of not greater than 100micrometers. For example, the average size can be not greater than 30micrometers, such as not greater than 1.5 micrometers, or not greaterthan 0.8 micrometers.

In an additional example of the seventh aspect or the above examples,the hydrophilic monomer includes an acrylamide monomer.

In a further example of the seventh aspect or the above examples, theparticles of the plurality of particles have an average porosity of atleast 60%.

In an eighth aspect, a composition includes an aqueous mixture of anacrylamide monomer and a crosslinker, the acrylamide monomer including ahydrophobic protection group, the monomer and crosslinker included in amass ratio of monomer:crosslinker in a range of 15:1 to 1:2.

In an example of the eighth aspect, the crosslinker is a divinylcrosslinker. For example, the divinyl crosslinker can include adiacrylamide. In another example, the diacrylamide includesN.N′-(ethane-1,2-diyl)bis(N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide,N,N′-(2-hydroxypropane-1,3-diyl)diacrylamide, a protected derivativethereof, or a combination thereof. The diacrylamide can, for example,includeN,N′-(ethane-1,2-diyl)bis(N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide,N,N′-(N-(2-((tert-butyldimethylsilyl)oxy)propane-1,3-diyl)diacrylamide,N,N′-(ethane-1,2-diyl)bis(N-(2-((triethylsilyl)oxy)ethyl)acrylamide,N,N′-(N-(2-((triethylsilyl)oxy)propane-1,3-diyl)diacrylamide,silyl-protected N-[2-(acryloylamino)-1,2-dihydroxyethyl]acrylamide suchas N,N′(2,3-bis((triethylsilyl)oxy)butane-1,4-diyl)diacrylamide, or acombination thereof. In an additional example, the divinyl crosslinkerincludes ethyleneglycoldimethacrylate, divinylbenzene, hexamethylenebisacrylamide, trimethylolpropane trimethacrylate, or a combinationthereof.

In another example of the eighth aspect or the above examples, the ratiois in a range of 10:1 to 1:1.

In a ninth aspect, a method of sequencing a polynucleotide includesproviding a device including an array of wells. At least one well isoperatively connected to an ISFET and includes a particle formed by themethod of the above aspects. The particle is attached to apolynucleotide. The method further includes applying a solutionincluding nucleotides of a predetermined type to the device andobserving an ionic response to the applying the solution.

In a tenth aspect, a method for nucleotide incorporation includesproviding a particle formed by the method of the above aspects. Theparticle is attached to a nucleic acid duplex including a templatenucleic acid hybridized to a primer. The duplex is bound to apolymerase. The method further includes contacting the particle with oneor more nucleotides and incorporating at least one nucleotide onto theend of the primer using the polymerase.

In an example of the tenth aspect, incorporating further includesgenerating a byproduct of nucleotide incorporation.

In another example of the tenth aspect and the above examples, themethod further includes detecting the incorporating by detecting thebyproduct using a field effect transistor (FET).

In an eleventh aspect, a method of forming a particle includes promotinga seed particle to form a disperse phase in an aqueous suspension, inthe disperse phase, polymerizing a plurality of mer units of ahydrophilic monomer having a hydrophobic protection group, therebyforming a polymeric particle including a plurality of hydrophobicprotection groups, and converting the polymeric particle to a hydrogelparticle.

In a twelfth aspect, a method of forming a particle includes providing aseed particle in an aqueous suspension, the seed particle comprising ahydrophobic polymer, and includes promoting the seed particle to form adisperse phase in the aqueous suspension. The method further includes,in the disperse phase, polymerizing a plurality of mer units of ahydrophilic monomer having a hydrophobic protection group, therebyforming a polymeric particle including a hydrophilic polymer having aplurality of the hydrophobic protection groups. The polymeric particleincludes the hydrophobic polymer. The method also includes cleaving theplurality of hydrophobic protection groups from the hydrophilic polymerand extracting the hydrophobic polymer from the polymeric particle toform a hydrogel particle.

In a thirteenth aspect, a particle includes a polymer formed frompolymerization of hydroxyalkyl acrylamide and a diacrylamide. Thediacrylamide includes a hydroxyl group. The particle absorbs at least300 wt % water based on the weight of the polymer when exposed to water.

In an example of the thirteenth aspect, the particle absorbs at least1000 wt % water based on the weight of the polymer when exposed towater.

In another example of the thirteenth aspect and the above examples, theparticle has a particle size is not greater than 100 micrometers. Forexample, the particle size can be not greater than 30 micrometers, suchas not greater than 1.5 micrometers.

In a further example of the thirteenth aspect and the above examples,the hydroxyalkyl acrylamide includes hydroxyethyl acrylamide.

In an additional example of the thirteenth aspect and the aboveexamples, the hydroxyalkyl acrylamide includesN-[tris(hydroxymethyl)methyl)acrylamide (A, illustrated below),N-(hydroxymethyl)acrylamide (B, illustrated below), or a combinationthereof.

In another example of the thirteenth aspect and the above examples, thediacrylamide includes N,N′-(ethane-1,2-diyl)bis(2-hydroxylethyl)acrylamide, N,N′-(2-hydroxypropane-1,3-diyl)diacrylamide, aprotected derivative thereof, or a combination thereof. The diacrylamidecan, for example, includeN,N′-(ethane-1,2-diyl)bis(N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide,N,N′-(N-(2-((tert-butyldimethylsilyl)oxy)propane-1,3-diyl)diacrylamide,N,N′-(ethane-1,2-diyl)bis(N-(2-((triethylsilyl)oxy)ethyl)acrylamide,N,N′-(N-(2-((triethylsilyl)oxy)propane-1,3-diyl)diacrylamide,silyl-protected N-[2-(acryloylamino)-1,2-dihydroxyethyl]acrylamide suchas N,N′(2,3-bis((triethylsilyl)oxy)butane-1,4-diyl)diacrylamide, or acombination thereof.

Embodiments may be in accordance with any one of the following numberedclauses.

1. A method of forming a particle, the method comprising: in a dispersephase within an aqueous suspension, polymerizing a plurality of merunits of a hydrophilic monomer having a hydrophobic protection group,thereby forming a polymeric particle including a plurality of thehydrophobic protection groups; and converting the polymeric particle toa hydrophilic particle.

2. The method of clause 1, wherein the hydrophilic particle is ahydrogel particle.

3. The method of clause 1 or clause 2, wherein the hydrophilic monomerincludes an acrylamide monomer.

4. The method of clause 1 or clause 2, wherein the hydrophilic monomeris a radically polymerizable monomer and the dispersed phase furtherincludes a diacrylamide crosslinker having a hydrophobic protectiongroup.

5. The method of any one of clauses 1-4, wherein converting thepolymeric particle includes removing at least a portion of the pluralityof the hydrophobic protection groups from the polymeric particle.

6. The method of clause 5, wherein removing at least a portion of theplurality of the hydrophobic protection groups includes acid cleaving atleast a portion of the plurality of the hydrophobic protection groupsfrom the polymeric particle.

7. The method of any one of clauses 1-6, wherein converting thepolymeric particle includes removing substantially all of the pluralityof hydrophobic protection groups from the polymeric particle.

8. The method of any one of clauses 1-7, further comprising promoting aseed particle in the aqueous suspension to form the dispersed phase.

9. The method of clause 8, wherein the mass ratio of protectedmonomer:seed particles is in a range of 150:1 to 1:1.

10. The method of clause 8, wherein the seed particle includes a seedpolymer.

11. The method of clause 10, further comprising extracting the seedpolymer after converting the polymeric particle.

12. The method of clause 10, wherein the seed polymer is hydrophobic.

13. The method of clause 10, wherein the seed polymer includes astyrenic polymer, an acrylic polymer, an acrylamide, another vinylpolymer, or a combination thereof.

14. The method of clause 8, wherein the seed particle has an initialparticle size of not greater than 0.6 micrometers.

15. The method of clause 14, wherein the initial particle size is notgreater than 0.45 micrometers.

16. The method of clause 15, wherein the initial particle size is notgreater than 0.35 micrometers.

17. The method of clause 16, wherein the initial particle size is notgreater than 0.15 micrometers.

18. The method of clause 8, wherein the seed particle has an initialparticle size in a range of 1 micrometer to 7 micrometers.

19. The method of clause 8, wherein promoting the seed particle includesmixing a solvent and a promoting agent with the seed particle.

20. The method of clause 19, wherein the promoting agent is hydrophobicand has a water solubility of less than 0.01 g/l at 25° C.

21. The method of clause 19, wherein the promoting agent includesdioctanoyl peroxide or dioctyladipate or polystyrene with molecularweight below 20 kD.

22. The method of any one of clauses 1-21, wherein the hydrophilicmonomer includes an acrylamide.

23. The method of any one of clauses 4-22, wherein the radicallypolymerizable monomer is a vinyl-based monomer.

24. The method of clause 23, wherein the vinyl-based monomer includes anacrylate, an acrylamide, a vinyl alcohol, a vinyl acetate,acrylamido-methyl-propanesulfonic acid, or a combination thereof.

25. The method of clause 24, wherein the vinyl-based monomer is anacrylamide.

26. The method of any one of clauses 1-25, wherein the hydrophobicprotection group includes a hydroxyl protection group.

27. The method of any one of clauses 1-25, wherein the hydrophobicprotection group includes an amine protection group.

28. The method of any one of clauses 1-27, wherein the hydrophobicprotection group includes an organometallic moiety.

29. The method of clause 28, wherein the organometallic moiety forms asilyl ether functional group.

30. The method of clause 29, wherein the silyl ether function group isderived from tert-butyldimethylsilane ether, trimethylsilyl ether,triethylsilyl ether, diphenyl methyl silyl ether, or a combinationthereof.

31. The method of any one of clauses 1-30, wherein polymerizing theplurality of mer units further includes mixing a crosslinker with thehydrophilic monomer having a hydrophobic protection group.

32. The method of clause 31, wherein mixing the crosslinker includesmixing the crosslinker at a mass ratio of hydrophilicmonomer:crosslinker in a range of 15:1 to 1:2.

33. The method of clause 32, wherein the range is 10:1 to 1:1.

34. The method of clause 31, wherein the crosslinker is a low watersolubility crosslinker and has a water solubility of less than 10 g/l at25° C.

35. The method of clause 31, wherein the crosslinker is a divinylcrosslinker.

36. The method of clause 35, wherein the divinyl crosslinker includes adiacrylamide.

37. The method of clause 4 or clause 36, wherein the diacrylamideincludesN,N′-(ethane-1,2-diyl)bis(N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide,N,N′-(2-hydroxypropane-1,3-diyl)diacrylamide, a protected derivativethereof, or a combination thereof.

38. The method of clause 4 or clause 37, wherein the diacrylamideincludesN,N′-(ethane-1,2-diyl)bis(N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide,N,N′-(N-(2-((tert-butyldimethylsilyl)oxy)propane-1,3-diyl)diacrylamide,N,N′-(ethane-1,2-diyl)bis(N-(2-((triethylsilyl)oxy)ethyl)acrylamide,N,N′-(N-(2-((triethylsilyl)oxy)propane-1,3-diyl)diacrylamide,silyl-protected N-[2-(acryloylamino)-1,2-dihydroxyethyl]acrylamide suchas N,N′(2,3-bis((triethylsilyl)oxy)butane-1,4-diyl)diacrylamide, or acombination thereof.

39. The method of clause 35, wherein the divinyl crosslinker includesethyleneglycoldimethacrylate, divinylbenzene, hexamethylenebisacrylamide, trimethylolpropane trimethacrylate, or a combinationthereof.

40. The method of any one of clauses 1-39, wherein polymerizing theplurality of mer units includes mixing a porogen with the hydrophilicmonomer having a hydrophobic protection group.

41. The method of clause 40, wherein the porogen is an aromatic porogen.

42. The method of clause 41, wherein the aromatic porogen includestoluene, xylene, mesitylene, phenylenethyl acetate or ethylbenzoate.

43. The method of any one of clauses 1-42, further comprising activatingthe hydrophilic particle or the hydrogel particle.

44. The method of clause 43, wherein converting includes providing oneor more hydroxyl groups on the hydrogel particle and wherein activatingincludes converting at least one of the one or more hydroxyl groups toalkyl or aryl sulfonic esters.

45. The method of clause 43, wherein converting includes providing oneor more hydroxyl groups on the hydrogel particle and wherein activatingincludes replacing at least one of the one or more hydroxyl groups withan azide functional moiety.

46. The method of clause 43, wherein converting includes providing oneor more amine groups on the hydrogel particle and wherein activatingincludes reacting at least one of the one or more amine groups withbis-succinimidyl C2-C12 alkyl ester.

47. The method of clause 43, further comprising binding anoligonucleotide to the activated hydrogel polymer.

48. The method of clause 47, wherein binding includes nucleophilicsubstitution and the oligonucleotide is a nucleophile-terminatedoligonucleotide.

49. The method of clause 48, wherein a nucleophile of thenucleophile-terminated oligonucleotide is an amine group.

50. The method of clause 48, wherein a nucleophile of thenucleophile-terminated oligonucleotide is a thiol group.

51. The method of clause 47, further comprising hybridizing apolynucleotide to the oligonucleotide.

52. The method of clause 51, further comprising amplifying thepolynucleotide into a plurality of polynucleotides and attaching atleast a portion of the plurality of polynucleotides to the hydrogelparticle, thereby generating a hydrogel particle including a pluralityof attached polynucleotides.

53. The method of clause 51, further comprising amplifying thepolynucleotide into a plurality of complementary polynucleotides byextending the oligonucleotide, thereby generating a hydrogel particleincluding a plurality of attached polynucleotides.

54. The method of any one of clauses 1-53, wherein the hydrogel particleis one of a plurality of similarly formed hydrogel particles having anaverage particle size of not greater than 2 micrometer in water.

55. The method of clause 54, wherein the average particle size is notgreater than 1 micrometer.

56. The method of clause 55, wherein the average particle size is notgreater than 0.8 micrometers.

57. The method of clause 56, wherein the average particle size is notgreater than 0.5 micrometers.

58. The method of any one of clauses 1-44, wherein the hydrogel particleis one of a plurality of similarly formed hydrogel particles having anaverage particle size in a range of 5 micrometers to 100 micrometers inwater.

59. The method of any one of clauses 1-58, wherein the hydrogel particleis one of a plurality of similarly formed hydrogel particles that aresubstantially uniform in size.

60. The method of any one of clauses 1-59, wherein the hydrogel particleis one of a plurality of similarly formed hydrogel particles having acoefficient of variance of not greater than 5.0%.

61. The method of clause 60, wherein the coefficient of variance of notgreater than 3.5%.

62. A method of forming a particle, the method comprising: polymerizinga plurality of mer units of a hydrophilic monomer having a hydrophobicprotection group, thereby forming a polymeric particle including aplurality of the hydrophobic protection groups; removing at least aportion of plurality of the hydrophobic protection groups from thepolymeric particle to form a hydrophilic particle; and binding anoligonucleotide to the hydrophilic particle.

63. The method of clause 62, wherein removing at least a portion of theplurality of the hydrophobic protection groups includes acid cleaving atleast a portion of the plurality of the hydrophobic protection groupsfrom the polymeric particle.

64. The method of clause 62 or 63, wherein the hydrophilic monomerincludes an acrylamide.

65. The method of any one of clauses 62-64, wherein the hydrophobicprotection group includes a hydroxyl protection group.

66. The method of any one of clauses 62-65, wherein the hydrophobicprotection group includes an organometallic moiety.

67. The method of clause 66, wherein the organometallic moiety forms asilyl ether functional group.

68. The method of any one of clauses 62-67, wherein polymerizing theplurality of mer units further includes mixing a crosslinker with thehydrophilic monomer having a hydrophobic protection group.

69. The method of clause 68, wherein the crosslinker is a divinylcrosslinker.

70. The method of clause 69, wherein the divinyl crosslinker includes adiacrylamide.

71. The method of any one of clauses 62-70, further comprisingactivating the hydrogel particle prior to binding the oligonucleotide.

72. The method of clause 71, wherein removing includes providing one ormore hydroxyl groups on the hydrophilic particle and wherein activatingincludes converting at least one of the one or more hydroxyl groups to asulfonate ester group.

73. The method of clause 71, wherein removing includes providing one ormore hydroxyl groups on the hydrophilic particle and wherein activatingincludes replacing at least one of the one or more hydroxyl groups withan azide functional moiety.

74. The method of clause 71, wherein binding includes binding theoligonucleotide to the activated hydrogel polymer.

75. The method of clause 74, wherein binding includes nucleophilicsubstitution and the oligonucleotide is a nucleophile-terminatedoligonucleotide.

76. The method of clause 75, wherein a nucleophile of thenucleophile-terminated oligonucleotide is an amine group.

77. The method of clause 75, wherein a nucleophile of thenucleophile-terminated oligonucleotide is a thiol group.

78. The method of any one of clauses 62-77, further comprisinghybridizing a polynucleotide to the oligonucleotide.

79. The method of clause 78, further comprising amplifying thepolynucleotide into a plurality of polynucleotides and attaching atleast a portion of the plurality of polynucleotides to the hydrogelparticle, thereby generating a hydrogel particle including a pluralityof attached polynucleotides.

80. The method of clause 78, further comprising amplifying thepolynucleotide into a plurality of complementary polynucleotides byextending the oligonucleotide, thereby generating a hydrogel particleincluding a plurality of attached polynucleotides.

81. A plurality of particles comprising at least 100,000 particles, atleast one particle of the plurality of particles comprising a hydrogel,the plurality of particles having an average particle size of notgreater than 100 micrometers and a coefficient of variance of notgreater than 5%.

82. The plurality of particles of clause 81, wherein each of the atleast 100,000 particles comprises the hydrogel.

83. The plurality of particles of clause 81 or 82, wherein thecoefficient of variance is not greater than 4.5%.

84. The plurality of particles of clause 83, wherein the coefficient ofvariance is not greater than 4.0%.

85. The plurality of particles of clause 84, wherein the coefficient ofvariance is not greater than 3.5%.

86. The plurality of particles of clause 85, wherein the coefficient ofvariance is not greater than 3.0%.

87. The plurality of particles of any one of clauses 81-86, wherein theaverage size is not greater than 30 micrometers.

88. The plurality of particles of clause 87, wherein the average size isnot greater than 1.5 micrometers.

89. The plurality of particles of clause 88, wherein the average size isnot greater than 1.1 micrometers.

90. The plurality of particles of clause 89, wherein the average size isnot greater than 0.6 micrometers.

91. The plurality of particles of clause 90, wherein the average size isnot greater than 0.5 micrometers.

92. The plurality of particles of any one of clauses 81-91, wherein thehydrogel includes an acrylamide polymer.

93. The plurality of particles of any one of clauses 81-92, wherein theparticles of the plurality of particles have an average porosity of atleast 60%.

94. A system comprising: an array of wells, at least one well of thearray of wells being operatively connected with an ISFET sensor; and aplurality of hydrogel particles having a coefficient of variance of notgreater than 5%, at least one of the hydrogel particles of the pluralityof hydrogel particles being disposed in a well of the array of wells.

95. A plurality of particles formed by the method comprising: in adisperse phase within an aqueous suspension, polymerizing a plurality ofmer units of a hydrophilic monomer having a hydrophobic protectiongroup, thereby forming a polymeric particle including a plurality of thehydrophobic protection groups; and converting the polymeric particle toa hydrogel particle.

96. The plurality of particles of clause 95, wherein the plurality ofparticles has a coefficient of variance of not greater than 5.0%.

97. The plurality of particles of clause 96, wherein the coefficient ofvariance is not greater than 4.0%.

98. The plurality of particles of clause 97, wherein the coefficient ofvariance is not greater than 3.5%.

99. The plurality of particles of clause 98, wherein the coefficient ofvariance is not greater than 3.0%.

100. The plurality of particles of any one of clauses 95-99, wherein theplurality of particles have an average size of not greater than 100micrometers.

101. The plurality of particles of clause 100, wherein the average sizeis not greater than 30 micrometers.

102. The plurality of particles of clause 101, wherein the average sizeis not greater than 5 micrometers.

103. The plurality of particles of clause 102, wherein the average sizeis not greater than 1.5 micrometers.

104. The plurality of particles of clause 103, wherein the average sizeis not greater than 0.8 micrometers.

105. The plurality of particles of any one of clauses 95-104, whereinthe hydrophilic monomer includes an acrylamide monomer.

106. The plurality of particles of any one of clauses 95-105, whereinthe particles of the plurality of particles have an average porosity ofat least 60%.

107. A composition comprising an aqueous mixture of an acrylamidemonomer and a crosslinker, the acrylamide monomer including ahydrophobic protection group, the monomer and crosslinker included in amass ratio of monomer:crosslinker in a range of 15:1 to 1:2.

108. The composition of clause 107, wherein the crosslinker is a divinylcrosslinker.

109. The composition of clause 108, wherein the divinyl crosslinkerincludes a diacrylamide.

110. The composition of clause 109, wherein the diacrylamide includesN.N′-(ethane-1,2-diyl)bis(N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide,N,N′-(2-hydroxypropane-1,3-diyl)diacrylamide, a protected derivativethereof, or a combination thereof.

111. The composition of clause 110, wherein the diacrylamide includesN,N′-(ethane-1,2-diyl)bis(N-(2-((tert-butyldimethylsilyl)oxy)ethyl)acrylamide,N,N′-(N-(2-((tert-butyldimethylsilyl)oxy)propane-1,3-diyl)diacrylamide,N,N′-(ethane-1,2-diyl)bis(N-(2-((triethylsilyl)oxy)ethyl)acrylamide,N,N′-(N-(2-((triethylsilyl)oxy)propane-1,3-diyl)diacrylamide,silyl-protected N-[2-(acryloylamino)-1,2-dihydroxyethyl]acrylamide suchas N,N′(2,3-bis((triethylsilyl)oxy)butane-1,4-diyl)diacrylamide, or acombination thereof.

112. The composition of clause 108, wherein the divinyl crosslinkerincludes ethyleneglycoldimethacrylate, divinylbenzene, hexamethylenebisacrylamide, trimethylolpropane trimethacrylate, or a combinationthereof.

113. The composition of any one of clauses 107-112, wherein the ratio isin a range of 10:1 to 1:1.

114. A method of sequencing a polynucleotide, the method comprising:providing a device including an array of wells, at least one well beingoperatively connected to an ISFET and including a particle formed by themethod of any one of clauses 1-80, the particle being attached to apolynucleotide, applying a solution including nucleotides of apredetermined type to the device; and observing an ionic response to theapplying the solution.

115. A method for nucleotide incorporation, comprising: providing aparticle formed by the method any one of clauses 1-80, the particlebeing attached to a nucleic acid duplex including a template nucleicacid hybridized to a primer, the duplex being bound to a polymerase;contacting the particle with one or more nucleotides; and incorporatingat least one nucleotide onto the end of the primer using the polymerase.

116. The method of clause 115, wherein the incorporating furtherincludes generating a byproduct of nucleotide incorporation.

117. The method of clause 115 or 116, further including detecting theincorporating by detecting the byproduct using a field effect transistor(FET).

118. A method of forming a particle, the method comprising: promoting aseed particle to form a disperse phase in an aqueous suspension; in thedisperse phase, polymerizing a plurality of mer units of a hydrophilicmonomer having a hydrophobic protection group, thereby forming apolymeric particle including a plurality of hydrophobic protectiongroups; and converting the polymeric particle to a hydrogel particle.

119. A method of forming a particle, the method comprising: providing aseed particle in an aqueous suspension, the seed particle comprising ahydrophobic polymer; promoting the seed particle to form a dispersephase in the aqueous suspension; in the disperse phase, polymerizing aplurality of mer units of a hydrophilic monomer having a hydrophobicprotection group, thereby forming a polymeric particle including ahydrophilic polymer having a plurality of the hydrophobic protectiongroups, the polymeric particle including the hydrophobic polymer;cleaving the plurality of hydrophobic protection groups from thehydrophilic polymer; and extracting the hydrophobic polymer from thepolymeric particle to form a hydrogel particle.

120. A population of particles having a coefficient of variance of notgreater than 5% and comprising a polymer formed from polymerization ofhydroxyalkyl acrylamide and a diacrylamide, the diacrylamide including ahydroxyl group, wherein the particle absorbs at least 300 wt % waterbased on the weight of the polymer when exposed to water.

121. The particle of clause 120, wherein the particle absorbs at least1000 wt % water based on the weight of the polymer when exposed towater.

122. The particle of clause 120 or 121, wherein the particle has aparticle size of not greater than 100 micrometers.

123. The particle of clause 122, wherein the particle size is notgreater than 30 micrometers.

124. The particle of clause 123, wherein the particle size is notgreater than 1.5 micrometers.

125. The particle of any one of clauses 120-124, wherein thehydroxyalkyl acrylamide includes hydroxyethyl acrylamide.

126. The particle of any one of clauses 120-125, wherein thehydroxyalkyl acrylamide includesN-[tris(hydroxymethyl)methyl)acrylamide, N-(hydroxymethyl)acrylamide, ora combination thereof.

127. The particle of any one of clauses 120-126, wherein thediacrylamide includes N,N′-(ethane-1,2-diyl)bis(2-hydroxylethyl)acrylamide, N,N′-(2-hydroxypropane-1,3-diyl)diacrylamide, aprotected derivative thereof, or a combination thereof.

Note that not all of the activities described above in the generaldescription or the examples are required, that a portion of a specificactivity may not be required, and that one or more further activitiesmay be performed in addition to those described. Still further, theorder in which activities are listed are not necessarily the order inwhich they are performed.

In the foregoing specification, the concepts have been described withreference to specific embodiments. However, one of ordinary skill in theart appreciates that various modifications and changes can be madewithout departing from the scope of the invention as set forth in theclaims below. Accordingly, the specification and figures are to beregarded in an illustrative rather than a restrictive sense, and allsuch modifications are intended to be included within the scope ofinvention.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having” or any other variation thereof, areintended to cover a non-exclusive inclusion. For example, a process,method, article, or apparatus that comprises a list of features is notnecessarily limited only to those features but may include otherfeatures not expressly listed or inherent to such process, method,article, or apparatus. Further, unless expressly stated to the contrary,“or” refers to an inclusive-or and not to an exclusive-or. For example,a condition A or B is satisfied by any one of the following: A is true(or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

Also, the use of “a” or “an” are employed to describe elements andcomponents described herein. This is done merely for convenience and togive a general sense of the scope of the invention. This descriptionshould be read to include one or at least one and the singular alsoincludes the plural unless it is obvious that it is meant otherwise.

Benefits, other advantages, and solutions to problems have beendescribed above with regard to specific embodiments. However, thebenefits, advantages, solutions to problems, and any feature(s) that maycause any benefit, advantage, or solution to occur or become morepronounced are not to be construed as a critical, required, or essentialfeature of any or all the claims.

After reading the specification, skilled artisans will appreciate thatcertain features are, for clarity, described herein in the context ofseparate embodiments, may also be provided in combination in a singleembodiment. Conversely, various features that are, for brevity,described in the context of a single embodiment, may also be providedseparately or in any subcombination. Further, references to valuesstated in ranges include each and every value within that range.

What is claimed is:
 1. A system comprising: an array of wells, at leastone well of the array of wells being operatively connected with an ISFETsensor; and a plurality of hydrogel particles having a coefficient ofvariance of not greater than 5%, at least one of the hydrogel particlesof the plurality of hydrogel particles being disposed in a well of thearray of wells.
 2. The system of claim 1, wherein the hydrogel particlescomprise a polymer formed from polymerization of hydroxyalkyl acrylamideand a diacrylamide, the diacrylamide including a hydroxyl group.
 3. Thesystem of claim 2, wherein the hydroxyalkyl acrylamide includeshydroxyethyl acrylamide.
 4. The system of claim 2, wherein thehydroxyalkyl acrylamide includesN-[tris(hydroxymethyl)methyl)acrylamide, N-(hydroxymethyl)acrylamide, ora combination thereof.
 5. The system of claim 2, wherein thediacrylamide includes N,N′-(ethane-1,2-diyl)bis(2-hydroxylethyl)acrylamide, N,N′-(2-hydroxypropane-1,3-diyl)diacrylamide, aprotected derivative thereof, or a combination thereof.
 6. The system ofclaim 1, wherein the hydrogel particles absorb at least 300 wt % waterbased on the weight of the polymer when exposed to water.
 7. The systemof claim 6, wherein the hydrogel particles absorb at least 1000 wt %water based on the weight of the polymer when exposed to water.
 8. Thesystem of claim 1, wherein the hydrogel particles have an averageparticle size of not greater than 100 micrometers.
 9. The system ofclaim 8, wherein the average particle size is not greater than 30micrometers.
 10. The system of claim 9, wherein the average particlesize is not greater than 1.5 micrometers.
 11. The system of claim 10,wherein the average size is not greater than 1.1 micrometers.
 12. Thesystem of claim 11, wherein the average size is not greater than 0.6micrometers.
 13. The system of claim 1, wherein the coefficient ofvariance is not greater than 4.5%.
 14. The system of claim 13, whereinthe coefficient of variance is not greater than 4.0%.
 15. The system ofclaim 14, wherein the coefficient of variance is not greater than 3.5%.16. The system of claim 15, wherein the coefficient of variance is notgreater than 3.0%.
 17. The system of claim 1, wherein the hydrogelparticles of the plurality of particles have an average porosity of atleast 60%.
 18. The system of claim 1, wherein the hydrogel particles areconjugated to polynucleotides.
 19. The system of claim 18, wherein thehydrogel particles have a nucleotide density of at least 10⁵ per μm³ andnot greater than 10¹⁵ per μm³.
 20. The system of claim 1, wherein theISFET sensor is operable to detect pH.